Microneedle-based transdermal delivery system and method of making same

ABSTRACT

A transdermal delivery system of microneedles containing a bioactive material, comprising at least one layer of a support material; at least one biodegradable needle associated with the support material, each needle comprising at least one biodegradable polymer and at least one sugar, wherein each biodegradable needle is hollow and is adapted to retain a bioactive material.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending applicationSer. No. 14/874,978, filed Oct. 5, 2015, entitled NanospheresEncapsulating Bioactive Material and Method for Formulation ofNanospheres, which claims benefit of priority to U.S. patent applicationSer. No. 12/569,867, filed Sep. 29, 2009, now U.S. Pat. No. 9,149,441,issued Oct. 6, 2015, and corresponding Provisional U.S. PatentApplication No. 61/100,886, filed Sep. 29, 2008, and commonly assignedto the assignee of the present application, the disclosure of which isincorporated by reference in its entirety herein.

FIELD

The present disclosure relates, in exemplary embodiments, to a systemfor delivering bioactive and other materials through the skin(transdermally) using microneedles containing the material. The presentdisclosure also relates, in exemplary embodiments, to methods forforming biodegradable microneedles containing the bioactive material.

BACKGROUND

Currently, most vaccines are administered via subcutaneous orintramuscular route. These have been highly effective in generatingprotective immune response, but they remain invasive, potentiallypainful and require a skilled professional for vaccination. In anattempt to minimize some of these issues scientists have explored thepotential of delivering vaccine antigens intradermally usingmicroneedles. Microneedles, as the name indicates, are microndiameter-sized needles, which upon insertion into the skin result infoimation of aqueous conduits forming a passage for the vaccine antigenstowards the immune-competent skin layers. Due to their short needlelength, they avoid contact with the nerve endings in the dermis thusremain to be a painless mode of immunization. Recently FDA approvedIntanza™ (by Sanofi Pasteur), an intradermal influenza vaccine thatincorporates a 1.5 mm needle attached to a pre-filled syringe loadedwith flu antigens. It has been shown to be efficacious when comparedwith an IM flu vaccine thus bringing a switch from hypodermic needles to“micro”-needles for immunizations. This opens a new avenue of vaccinedelivery through an effective, painless and patient-friendly route ofadministration. However, heretofore, there has not been a biodegradableand biocompatible microneedle transdermal delivery system.

SUMMARY

The following presents a simplified summary in order to provide a basicunderstanding of some aspects of various invention embodiments. Thesummary is not an extensive overview of the invention. It is neitherintended to identify key or critical elements of the invention nor todelineate the scope of the invention. The following summary merelypresents some concepts of the invention in a simplified form as aprelude to the more detailed description below.

In exemplary embodiments, disclosed is a transdermal delivery system ofmicroneedles containing a bioactive material, comprising at least onelayer of a support material, at least one biodegradable needleassociated with the support material, each needle comprising at leastone biodegradable polymer and at least one sugar, wherein eachbiodegradable needle is hollow and is adapted to retain a bioactivematerial.

In exemplary embodiments, disclosed is a biodegradable microneedle,comprising: at least one biodegradable needle associated with thesupport material, each needle comprising at least one biodegradablepolymer, and at least one sugar; wherein each biodegradable needle is atleast partially hollow and is adapted to retain a bioactive material.

In exemplary embodiments, disclosed is a method of forming transdermaldelivery system, comprising: (a) providing at least one biodegradablepolymer material; (b) dissolving the polymer material in a solvent toform a solution; (c) mixing the solution of polymer material of step (b)with at least one sugar to form a polymer-sugar mixture; (d) providing abioactive material; (e) providing a microneedle mold; (f) adding thebioactive material and the polymer-sugar mixture of step (c) to themicroneedle mold; and, (g) forming at least one microneedle from thepolymer-sugar mixture, the at least one microneedle having at least aportion that is hollow, wherein the bioactive material is retainedwithin the hollow portion of the microneedle.

In exemplary embodiments, disclosed is a method of transdermallydelivering a bioactive material, comprising: (a) forming at least onebiodegradable and at least partially hollow microneedle from at leastone biodegradable polymer and at least one sugar; (b) associating abioactive material with the at least one microneedle; (c) associatingthe at least one microneedle with a backing layer; and, (d) contactingthe at least one microneedle containing the bioactive material with theskin of a subject, whereby the at least one microneedle introduces thebioactive material to the subject and the at least one microneedlebiodegrades.

In exemplary embodiments, disclosed is a method of forming transdeiiiialdelivery system, comprising: (a) mixing PVA, HPMC, and the at least onesugar in a vessel; (b) dissolving the mixture of step (a) in water toand mixing to form a mixture; (c) adding ammonium hydroxide to themixture of step (b) and mixing; (d) adding to the mixture of step (c) atleast one bioactive material in microencapsulated form to foim aformulation; (e) adding an aliquot of the formulation of step (d) to amicroneedle mold; and, (f) centrifuging the microneedle mold andformulation of step (e) to force the formulation into the microneedlemold.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings disclose exemplary embodiments in which like referencecharacters designate the same or similar parts throughout the figures ofwhich:

Example 1 Figures:

FIG. 1A is a diagram showing mechanisms of interaction of N. gonorrhoeaewith cells of the immune system.

FIG. 1B is a schematic diagram of a microparticulate delivery system.

FIG. 2 is schematic view of a microneedle device according to oneexemplary embodiment used for transdemial delivery of particles.

FIG. 3A is a scanning electron microscopic (SEM) image of formalin fixedwhole cell N. gonorrhea which is the antigen for the vaccine.

FIG. 3B is a SEM image of spray dried microparticles containing thegonorrhea vaccine antigen.

FIG. 3C is a SEM image of the dissolving microneedles which contain thegonorrhea vaccine microparticles for transdermal delivery.

FIG. 4 is a graph of N. Gonorrhea specific IgG antibody levelmeasurements in serum via ELISA.

FIG. 5 is a schematic flow diagram of one exemplary embodiment of asolvent casting process for fabrication of microneedles.

FIG. 6A is a photomicrograph of porcine ear skin sample showingmicrochannels created by a microneedles patch.

FIG. 6B is a photomicrograph of porcine ear skin sample showingmicrochannels created by a microneedles patch.

FIG. 7 is a schematic view of in vivo tracking over time (30 min., 3hours, 6 hours and 24 hours) of a vaccine after delivery to a mouse.

FIG. 8 is a SEM image of the microneedles being dissolved in the miceafter 12 minutes of application of the patch.

FIG. 9 is graph of serum IgG levels over time.

FIG. 10A is a graph of CD4 T cell counts of lymph nodes.

FIG. 10B is a graph of CD4 T cell counts of spleens.

FIG. 10C is a graph of CD8 T cell counts of lymph nodes.

FIG. 10D is a graph of CD8 T cell counts of spleens.

FIG. 11 is a graph of antigen specific CD4 and CD8 T cells counts.

Example 2 Figures:

FIG. 12A is a SEM image of polymer microparticle matrix formed.

FIG. 12B is a SEM image of breast cancer nano-vaccine particles.

FIG. 13 is a graph of induction of nitric oxide release by dendriticcells (DC 2.4).

FIG. 14A is a graph of MHC I expression.

FIG. 14B is a graph of CD 40 expression.

FIG. 14C is a graph of MHC II expression.

FIG. 14D is a graph of CD 80 expression.

FIG. 15 is a graph of tumor volume measurement.

FIG. 16A is a graph of percent cell count measurement of lymph nodes CD4 T cells.

FIG. 16B is a graph of percent cell count measurement of spleen CD 4 Tcells.

FIG. 16C is a graph of percent cell count measurement of spleen CD 8 Tcells.

FIG. 16D is a graph of percent cell count measurement of spleen CD 8 Tcells.

Example 3 Figures:

FIG. 17 is a schematic illustration of the structure of influenza virus.

FIG. 18 is transmission electron microscopy (TEM) image ofmicroparticles of inactivated influenza virus.

FIG. 19 is a schematic flow diagram of one exemplary embodiment of aprocess for fabrication of microneedles.

FIG. 20 is a graph of the measurement of mean fluorescent intensity ofsolutions and microparticles of various groups.

FIG. 21 is a graph of the measurement of mean fluorescent intensity ofsolutions and microparticles of various groups.

FIG. 22 is a graph of the measurement of mean fluorescent intensity ofsolutions and microparticles of various groups.

FIG. 23 is a graph of the measurement of mean fluorescent intensity ofsolutions and microparticles of various groups.

FIG. 24 is a graph of serum titers over time.

FIG. 25 is a graph of flow cytometry cell count of CD4 and CD8.

Example 4 Figures:

FIG. 26 is a schematic illustration of antigens and other materials in apolymer matrix.

FIG. 27 is a SEM image of microparticles.

FIG. 28 is a graph of nitric oxide concentration of various solutions.

FIG. 29 is a graph of mean fluorescence intensity of CD86 and CDE 80.

FIG. 30 is a graph of mean fluorescence intensity of CD40 and MHC II.

FIG. 31 is a graph of log titer over 32 weeks.

FIG. 32 is a graph of cell surface expression.

FIG. 33 is a graph of cell surface expression.

Example 5 Figures:

FIG. 34 is a SEM of vaccine loaded microparticles taken using the PhenomDesktop SEM by placing microparticles on a carbon film and observing at20kV. Microparticles are irregularly shaped in the size range of 1-2 um.

FIG. 35 is a photograph of SDS PAGE of F-VLPs extracted from microparticles. Samples were resolved on a prepared 12% polyacrylamide geland stained using Coomassie Blue. Lane 1: Molecular weight ladder (kDa);Lane 2: Blank microparticles (MPs); Lane 3: VLP-Suspension and Lane 4:F-VLP extracted from microparticles. The VLP was found to be intact inmicroparticulate form.

FIG. 36 is a graph showing the amount of nitric oxide released (μM) fromDC 2.4 cells when exposed to Cells Only, Blank MP, RSV F-VLP Suspension,RSV F-VLP MP (*p<0.05).

FIG. 37 is a graph showing the amount of nitric oxide released from DC2.4 cells when exposed to VLP Suspension, RSV VLP MP and RSV VLPMP+Alum, MPL A and MF59 (*p<0.05) There was a significant release ofnitric oxide seen in supernatant of cells receiving RSV F-VLP+Alum/ MF59MPs.

FIG. 38 is a graph showing the expression of CD40 on DC 2.4 cellsexposed to Blank MP, F-VLP solution, F-VLP MP and VLP MP with adjuvant.MHC II expression was significantly higher in MP group compared to VLPsolution.

FIG. 39 is a graph showing the expression of CD80 on DC 2.4 cellsexposed to Blank MPs, F-VLP solution, F-VLP MP and VLP MP withadjuvants. CD80 is a co-stimulatory molecule required for activation ofCD8 T cells.

FIG. 40 is a graph showing the expression of CD40 on DC 2.4 cellsexposed to Blank MP, F-VLP solution, F-VLP MP and VLP MP with adjuvant.

FIG. 41 is a schematic illustration of a timeline for an animal study.

FIG. 42 is a graph showing IgG antibody levels in blood serum of miceinoculated with Inactivated RSV vaccine (FI-RSV), solution form ofF-VLP, F-VLP microparticles and F-VLP+MPL microparticles.

FIG. 43 is a graph showing body weight measurements of mice 6 dayspost-challenge with live RSV A2 virus. Untreated mice (PBS) showed thehighest change in weight compared to vaccinated mice.

FIG. 44 is a graph of (spleen) CD4+ and CD8+ T cell response afterchallenge with live RSV A2 virus.

FIG. 45 is a graph of (lymph node) CD4+ and CD8+ T cell response afterchallenge with live RSV A2 virus.

FIG. 46 is a graph of viral titers measured in lung homogenates ofvarious groups after challenge using RT-PCR.

Example 6 Figures:

FIG. 47 is a schematic flow diagram of one exemplary embodiment of amethod used for the preparation of microcapsules encapsulating livepancreatic beta cells using biocompatible polymer.

FIG. 48 is a schematic diagram showing a portion of an exemplaryembodiment of a method of forming microcapsules.

FIG. 49 is a graph of the size of microcapsules (diameter being in μm)plotted at different gas flow rates. Plotted values are mean with±standard deviation bars.

FIG. 50 is a graph of microcapsule size distribution obtained afterspraying the alginate suspension at 250 L/Hr.

FIG. 51 are microcapsule images taken at 10× after spraying the alginatesuspension at 250 L/Hr.

FIG. 52 is a graph of FTIR spectra of sodium alginate.

FIG. 53 is a graph of FTIR spectra of alginate microcapsules.

FIG. 54A is a light microscopic image showing clusters of microcapsulesencapsulated beta islet pancreatic cells were taken at magnification of10×.

FIG. 54B is a light microscopic image showing clusters of microcapsulesencapsulated beta islet pancreatic cells were taken at magnification and40×.

FIG. 55 is a chart of Live dead cell stained images of microcapsulesencapsulated pancreatic islet beta cells collected over thirty daysperiod at magnification of 10×.

FIG. 56A is a graph of nitric oxide release vs MC Blank (Microcapsuleswithout beta islet pancreatic cells), MC cells (Microcapsulesencapsulate beta islet pancreatic cells and Cells only (Unencapsulatedcells) ns-not significant, **p<0.01very significant, ***p<0.001extremely significant.

FIG. 56B is a graph of nitric oxide release vs MC Blank (Microcapsuleswithout beta islet pancreatic cells), MC cells (Microcapsulesencapsulate beta islet pancreatic cells and Cells only (Unencapsulatedcells) ns-not significant, **p<0.01very significant, ***p<0.001extremely significant.

FIG. 57 is graph of short term stability monitored by measuring thefraction of intact microcapsules at different concentration of chitosanused as second layer on alginate microcapsules.

FIG. 58 is a graph of long term stability monitored by measuring thefraction of intact microcapsules.

FIG. 59 is a graph of blood glucose levels of different groups in micemeasured for 35 days.

FIG. 60 is a graph of percent graft survival plotted for differentgroups i.e., Diabetic control, MC cells (Microcapsules encapsulatedpancreatic beta islet cells) and Cells only (Unencapsulated cells).

FIG. 61 is a graph of fractional weight of mice in different groupsmeasured for 35 days.

FIG. 62A is a graph of percentage of CD4 and CD8 positive cells plottedfor different groups i.e., Diabetic control, MC beta and naked CellsOnly. *p<0.05 significant, **p<0.01 very significant, ***p<0.001extremely significant (spleen cells).

FIG. 62B is a graph of percentage of CD4 and CD8 positive cells plottedfor different groups i.e., Diabetic control, MC beta and naked CellsOnly. *p<0.05 significant, **p<0.01 very significant, ***p<0.001extremely significant (spleen cells).

FIG. 63A is a graph of percentage of CD4 positive cells plotted fordifferent groups i.e., Diabetic control, MC beta and naked Cells Only.*p<0.05 significant, **p<0.01very significant, ***p<0.001 extremelysignificant (Lymph node cells).

FIG. 63B is a graph of percentage of CD8 positive cells plotted fordifferent groups i.e., Diabetic control, MC beta and naked Cells Only.*p<0.05 significant, **p<0.01very significant, ***p<0.001 extremelysignificant (Lymph node cells).

FIG. 64 is a graph of a flow cytometric analysis showing CD45R cellcounts in different groups of mice *p<0.05 significant, **p<0.01verysignificant, ***p<0.001 extremely significant.

FIG. 65 is a graph of a flow cytometric analysis showing CD62L cellcounts in different groups of mice *p<0.05 significant, **p<0.01verysignificant, ***p<0.001 extremely significant.

DETAILED DESCRIPTION

While transdermal delivery through skin is referred to herein, thepresently disclosed microneedle systems and methods can be adapted fordelivery to or through other structures, such as, but not limited to,blood vessel walls, muscle tissue, organs, and the like.

The following examples are set forth for purposes of illustration only.Parts and percentages appearing in such examples are by weight unlessotherwise stipulated.

EXAMPLE 1

Transdermal Microneedle Based Particulate Vaccines

1) Transdemial Microneedle Based Particulate Neisseria gonorrheaeVaccine

BACKGROUND AND SIGNIFICANCE

Neisseria gonorrhoeae (the gonococcus, or GC) remains an importantdisease. Still relatively common in the US, with over 300,000 reportedcases annually, and probably as many that are not reported, it is muchmore common in Africa and in many other parts of the less-developedworld (Ison, Deal, & Unemo, 2013). Untreated gonococcal infection inwomen may progress to pelvic inflammatory disease, increasing the riskof ectopic pregnancy and infertility (Edwards, Jennings, Apicella, &Seib, 2016). The main reason to urge development of a gonococcal vaccineis emergence of antibiotic resistant GC. In many parts of the world,fluorinated quinolones are no longer recommended because of theprevalence of resistance (Jerse & Deal, 2013). The increasing threat ofdifficult-to-treat GC should make a gonococcal vaccine an importantobjective (Tapsall, 2009).

The Gc interacts with the immune system and prevents the generation ofan adaptive immune response. Gc can interact with various immune cellsto elicit innate inflammatory responses and suppress Th1/Th2-mediatedspecific immune responses (FIG. 1). (A) Phagocytosis by macrophagesresults in activation of NLRP3 inflammasomes, the production of IL-1 andactivation of PMNs, and activation of cathepsin B, which leads topyronecrosis of APC (Duncan et al., 2009). (B) Interactions with DCslead to up-regulation of PDL-1 and PDL-2, which induce apoptosis ofcells bearing PD1. This up-regulation also causes release of IL-10,which has immunoregulatory properties and stimulates type 1 regulatory Tcells (Tr1) (Zhu et al., 2012). (C)Interaction with CD4+ T helper cells(or B cells) induces secretion of IL-10, TGF-β, and IL-6 (Liu, Islam,Jarvis, Gray-Owen, & Russell, 2012). IL-10 and TGF-β suppress theactivation of Th1 and Th2 cells both directly, and through theactivation of Tr1 cells. TGF-β and IL-6 drive the development of Th17cells which secrete IL-17 and IL-22, leading to the recruitment orinduction of innate defenses such as PMNs and anti-microbial peptides.Gc is able to resist destruction by PMNs and anti-microbial peptideswhile concomitantly suppressing the development of adaptive immuneresponses such as Ge-specific antibodies that could enhance phagocytosisand intracellular killing by phagocytes and bacteriolysis through theclassical complement pathway (Jerse, Bash, & Russell, 2014). Thusexploring a transdermal microparticle vaccine formulation that confersprotection in patients, induces herd immunity, and provides significantadvantages over the conventional antibiotic therapy will have highpublic health impact in the United States.

Specific Aims:

Specific Aim 1: Optimize/characterize a novel whole-cell inactivatedNeisseria gonorrheae nanovaccine formulation delivered usingbiodegradable microneedle skin patch:

1. Determine the optimal dose of antigen encapsulation intonanoparticles for further loading into biodegradable microneedles

2. Characterize the novel gonococcal nanoparticles formulation long-termstability, antigens release and antigens uptake by antigen presentingcells (macrophages and dendritic cells) in vitro.

3. Effective antigen presentation by APCs or dendritic cells pulsed withgonococcal nanoparticles (measure co-stimulatory molecules induction,DCs maturation markers and T cell proliferation

4. Monitor in vivo subcutaneous antigen release from gonococcalnanoparticles loaded into microneedles skin patch in mice usingfluorescent probe bound to nanoparticles and imaging method. Monitorduration and scale of antigen release as nanoparticles diffusion intosubcutaneous skin.

Specific Aim 2: Determine the correlates of protection in immunizedmice:

1. Compare gonococcal nanoparticles-loaded into microneedles skin patchto gonococcal nanoparticles vaccine injected intraperitoneally in mice.

2. Measure the induction of antibody titers, total IgG and subclasseselicited against gonococcal antigens in vaccinated mice sera, andantigen-specific CD4/CD8 T cell in spleens and lymph nodes of vaccinatedmice.

3. Determine the serum opsonic and bactericidal activity of elicitedantibodies against live Neisseria gonorrheae using the parent strain F62as well as other clinical gonococcal isolates (cross protection).

Innovation

A. Microparticulate Vaccines

We have developed a biodegradable and biocompatible polymer matrixsystem for making the microparticles loaded with the vaccine using thespray drier method (Chablani et al., 2012; Shastri, Kim, Quan, D'Souza,& Kang, 2012; Ubale, D'Souza, Infield, McCarty, & Zughaier, 2013; Ubale,Gala, Zughaier, & D'Souza, 2014a). This method does not require organicsolvents which enhances the safety of such formulation. We formulated amicroparticle gonococcal vaccine that serves as a sustained releasesystem. The proposed vaccine formulation consists of formalin fixed deadwhole cell gonococcal encapsulated in albumin-based microparticles thatmimic the chemical conjugation process of CPS to a protein carriersimilar to meningitis, and enhance antigen uptake via albumin receptors,and elicit a T-cell-dependent immune response. Further, the proposedvaccine exists as a dry powder form and kept well protected frommoisture. Thus, the shelf lives of these vaccines are expected to beseveral fold higher than that of the conventional vaccines. The novelnanotechnology-based vaccine that mimics conjugation effects byencapsulation into albumin-based nanoparticle matrices provides thefollowing advantages: 1—does not require chemical conjugation, 2—selfadjuvanting-antigen delivery vehicle, 3—enhanced uptake by immune cellsand slow antigen release, i.e. antigen depot effect, 4—induces robustautophagy formation that enhances antigen presentation, 5—uses aheat-stable formulation that does not require refrigeration, 6—can beadministered via microneedles 7—the cost of producing this vaccine isdramatically reduced due to the elimination of the costs of chemicalconjugation process, purification and packaging in individual doseampoules that requires constant cooling with limited shelf life, and8—can be used to formulate and deliver other bacterial and viralvaccines.

B. Whole Cell Vaccine Antigen

Neisseria gonorrhoeae is the main bacteria which causes gonorrheainfections. There have been various approaches which have beeninvestigated for a vaccine strategy against gonorrhea infections. Sincethe immune suppression caused by the bacteria via various mechanismsexplained in FIG. 1 there was no adaptive immune response generated.Moreover there has not been much research on development of gonorrheavaccine which is evident from a PubMed search on Dec. 27, 2010 under“gonococcal vaccine” yielded 247 entries, whereas a similar search under“meningococcal vaccine” yielded 3326 entries. With the development ofantibiotic resistant strains of gonorrhea, FDA and CDC have prioritizedthe research on development of a vaccine against gonorrhea. We havedeveloped a novel approach where the whole cell of N. gonorrhea isformalin fixed for overnight and used as the vaccine antigen. By usingthe whole cell as the antigen, we preserve and present all the possibleantigenic proteins in their native form to the antigen presenting cells.This approach will cover all the antigenic sites and help in inducing animmune response. Moreover when encapsulated in a microparticulate form,it will be better up taken by the APCs and processed.

Microneedle Based Transdermal Vaccination:

The skin provides a unique site for the vaccination purposes as it iseasily accessible and houses various immune cells for an efficientimmune response against a range of antigens. Skin serves as a barrieragainst various pathogens and is equipped with the skin associatedlymphoid tissues (SALT) to combat any insult from invading pathogens.Various skin cells assist in generation of effective immune response(Gao, Pan, Chen, Xue, & Li, 2008). Keratinocytes are the mostpre-dominant (95%) epidermal cells in the skin. They can be activated bypathogens and result in production of cytokines, which in turn recruitsdendritic cells/antigen-presenting cells to the site of action leadingto initiation of the immune response. Skin host's a special kind ofdendritic cells, namely, the Langerhans cells. Langerhans cells compriseof only 2% of the total cell population in the epidermis but due totheir extended dendrites spread in the epidermal layer they cover over25% of the skin surface. These are professional phagocytic cellsefficient in immune surveillance and further signaling to the T-cellspresent in their vicinity. Activated macrophages and T-cells drain intonearby lymph nodes leading to an enhanced immune response. Currently,most of the vaccines are administered via subcutaneous or intramuscularroute. These have been highly effective in generating protective immuneresponse but they remain to be invasive, painful and require a skilledprofessional for vaccination. In an attempt to minimize some of theseissues scientists have explored the potential of delivering vaccineantigens intradermally using microneedles. Microneedles, as the nameindicates, are micron-sized diameter needles, which upon insertion intothe skin result in foiiiiation of aqueous conduits foiming a passage forthe vaccine antigens towards the immune-competent skin layers (FIG. 2).Due to their short needle length, they avoid contact with the nerveendings in the dermis thus remain to be a painless mode of immunization.Recently FDA approved IntanzaTM (by Sanofi Pasteur), an intrademialinfluenza vaccine that incorporates a 1.5 mm needle attached to apre-filled syringe loaded with flu antigens. It has been shown to beefficacious when compared with an IM flu vaccine thus bringing a switchfrom hypodermic needles to “micro”-needles for immunizations. This opensa new avenue of vaccine delivery through an effective, painless andpatient-friendly route of administration. The success of immunizationvia skin using microneedles inspired us to evaluate the potential ofdelivering gonorrhea vaccine through this route.

To summarize, multiple approaches were applied to ensure development ofpotent and efficacious vaccine against gonorrhea. Following innovativeapproaches will be combined:

1. Currently there is no approved FDA vaccine against gonorrheainfection. Development of an efficacious vaccine will have a majorimpact in combating the infection caused by drug resistant strains of N.Gonorrhea.

2. The entire cell surface of the N. Gonorrhea bacteria is preservedwhich helps to present all the antigenic sites on the bacterial surfaceto the immune system.

3. Particulate nature of vaccine ensures sustained release of antigens,higher internalization and stronger immune response than vaccinesolution

4. Transdermal immunization via biodegradable microneedles that rapidlydissolve or biodegrade in contact with water is a unique route ofadministration and thus exposing the antigens to the Langerhans cells,dermal dendritic cells.

5. The transdermal route offers a painless, self-administration andpatient compliant immunization strategy

6. We recently demonstrated the potential for a gonorrhea vaccine in amurine model (see preliminary data section).

Paradigm Shift:

Infections caused by Neisseria gonorrhoeae (the gonococcus) continue tobe a global, intractable problem. The absence of a gonococcal vaccine,together with the continuing emergence of antibiotic-resistant anduntreatable gonococcal strains, has raised awareness that N. gonorrhoeaeposes an “urgent” public health threat for which immediate aggressiveaction is greatly needed (Unemo & Shafer, 2014). Our approach of usingmicroparticulate based delivery system is believed to interact with theimmune system differently and has shown positive results in variousstudies. The particulate nature of the vaccine allow the vaccine antigento be taken by the dendritic cells and macrophages and thus enhance itspresentation and subsequent activation of T cells. Thus by using thewhole cell of gonorrhea in a microparticulate delivery system, and alsoharnessing the rich immune system of the skin, we believe to have apotential immunization strategy against the infections caused by N.Gonorrhea. We have got encouraging results for using this strategy (seepreliminary results) and believe this to be a potential vaccine againstgonorrhea.

Approach

Preliminary Results

Formulation of whole cell gonorrhea microparticulate vaccine: The N.gonorrhea cells were grown in culture medium. When confluent, the mediawas removed and a 10% solution of formalin was added and kept overnight.This will lead to fixation of the cells in their native form which wereused as the antigen for the vaccine (FIGS. 3A-B). FIGS. 3A-B shows thatthe cells were intact and were in their native form. This antigen wasmixed with a blend of biodegradable and biocompatible cellulose polymermatrix. This was then spray dried and microparticles were made. Thesemicroparticles were characterized for their size, charge etc.

Microparticulate Morphology and Characterization

Scanning electron microscopy (SEM) was performed to evaluate themicroparticle size and surface morphology. Microparticles were mountedonto metal stubs using double sided adhesive tape. After being coatedwith a thin layer (100-150° A), the microparticles were examined using aPhenom scanning electron microscope (FIGS. 3A-B).Recovery yield of themicroparticles after spray drying was calculated for all the formulatedbatches. Percent recovery yield was evaluated using the followingfoiriiula:

${{Percentage}\mspace{14mu} {Recovery}\mspace{14mu} {Yield}} = \frac{{Weight}{\mspace{11mu} \;}{of}\mspace{14mu} {microparticles}\mspace{14mu} {after}\mspace{14mu} {spray}{\mspace{11mu} \;}{drying}*100}{{Weight}\mspace{14mu} {of}\mspace{14mu} {all}{\mspace{11mu} \;}{ingredients}{\mspace{11mu} \;}{before}\mspace{14mu} {spray}\mspace{14mu} {drying}}$

Particle size of the optimized formulation was evaluated using theSpectrex Laser Particle counter that works on the principle of laserdiffraction. Particle size was measured in triplicates for blank as wellas vaccine microparticles. For zeta potential measurement, fivemicrograms of microparticles were suspended in 1 ml of deionized waterand measured using a Malvern Zetasizer. Zeta Potential was measured forblank as well as antigen loaded microparticles in triplicates. Theparticle size, recovery yield and zeta potential are all shown in Table1.

TABLE 1 Average Size 3.5 μm ± 1.2 μm Zeta Potential 7.1 mV ± 1.4 mVPercentage Yield 85%

Microparticles may be formed according to the method disclosed in U.S.Pat. No. 9,149,441 entitled NANOSPHERES ENCAPSULATING BIOACTIVE MATERIALAND METHOD FOR FORMULATION OF NANOSPHERES, the disclosure of which isincorporated herein in its entirety.

In one exemplary embodiment, microparticles can be prepared byspray-drying an aqueous suspension containing a bioactive material(e.g., whole cell lysate (WCL)), ethyl cellulose, cellulose acetatephthalate (CPD), hydroxyl-propyl methylcellulose acetate succinate(HPMCAS) and trehalose using the following formula: Whole cell lysateWCL=10% w/w, Ethyl cellulose=35% w/w, Cellulose acetate phthalate(CPD)=25% w/w, Hydroxyl-propyl methylcellulose acetate succinate(HPMCAS)=25%, and Trehalose=5% w/w. This final mixture was then spraydried using Buchi 290 Mini Spray Dryer (Buchi Corporation, Newcastle,Delaware) with an inlet temperature of 125° C. and outlet temperature of80° C. The particles were stored at −20° C. until further use. Inexemplary embodiments, microparticles including an adjuvant (that canenhance the immunogenicity of a vaccine) can be prepared following thesame procedure. In exemplary embodiments, particles may be made withadjuvant loading of 2.5% w/w.

In exemplary embodiments, the average particle size may be in a range ofabout 0.01-50 μm.

In exemplary embodiments, any of a variety of different sugars can beuses, including, but not limited to, trehalose, maltose, sucrose, or thelike.

Method

Dissolving microneedles, intended for the painless transdermal releaseof encapsulated pharmaceutical agents after dermal insertion, weredeveloped as a solution to the safety issue. Dissolvable microneedlesmainly deploy PDMS micromolds which are made from a master structure ofmicroneedles (FIG. 5). Briefly, Polydimethylsiloxane (PDMS) (DowChemicals) was poured onto the stainless steel master structure (Step1-3; FIG. 5). The microneedles were made using the following formula:

Drug/Particles=10% w/w (5 mg), Trehalose=25% w/w (12.5 mg), Maltose=25%w/w (12.5 mg), PVA=20% w/w (10 mg), HPMC=20% w/w (10 mg)

The calculated quantities in bracket are for 50 mg which is used formaking 2 microneedle patch). PVA, hydroxypropyl methyl cellulose (HPMC),Maltose and Trehalose were added to a 1.7 mL microcentrifuge tube. Thecontents were dissolved in minimum possible amount of water (e.g., 200mg of total solid content can be dissolved in 600 uL water). Vortex thecentrifuge tube. Then approximately 1/5th quantity (of water that wasadded to dissolve the solids) of Ammonium hydroxide (NH4OH) was added tothe microcentrifuge tube (here, 120 uL) and vortex again. The tube iskept aside for some time and observed if the contents are dissolved. Ifeverything goes into the solution, add the weighed amount of gonorrheavaccine microparticles in the end. This formulation is then added tomold avoiding air bubbles. These molds are then placed straight in 50 mLcentrifuge tubes. Centrifugation is done in the fixed angle centrifugein order to remove air bubbles and to force the formulation to go intothe microneedles mold. The maximum speed is 2000 rpm which is achievedstep wise, in order to avoid jerk in the rotation process, time forwhich centrifugation should be done is 5-10 min. Speed should be loweredgradually for same reason as above. After this centrifugation step, moreformulation is added to molds and centrifugation is repeated in the samemanner (Step 3-6; FIG. 5). This step can be repeated further by addingmore formulation or a blank backing layer solution. In exemplaryembodiments, the backing layer may be composed of the same components asthe microneedle matrix as described in various embodiments herein,however, the backing layer is formed without any microparticles. Keepthe molds in tubes and place it in an incubator at 37° C. overnight(Step 7-9; FIG. 5). The microneedle patch is 1 cm×1 cm in size whichcontains 100 microneedles (10 x 10). The microneedles are 600 μm.

In-vivo and In-Vitro Characterization of the Microneedles.

Scanning electron microscopy (SEM) was performed to evaluate themicroparticle size and surface morphology (FIG. 3C). The in-vitrodelivery of the microneedles was checked on a porcine ear skin sample(FIGS. 6A-B). In-order to track the vaccine and microparticles from themicroneedles, we prepared ICG loaded (IR dye) microneedles andadministered to the mice on the back and monitored the intensity of theIR dye at regular intervals (FIG. 7). We could see that the intensity ofthe dye after 30 mins of application was localized and around the siteof administration. As we monitored further, it spread to differentorgans in the mice at 6, 12 and 24 hours as shown in FIG. 7. In adifferent experiment to check the dissolution and delivery ofmicroneedles when applied to the skin, we dosed a microneedle patch to amice and hold it with an adhesive backing layer for 10-15 minutes. Thenthe microneedles patch was removed and SEM was taken to check if themicroneedles and dissolved. From FIG. 8, we can see that after 12minutes of application of the microneedles patch, all the needles haddissolved and only the back layer was left. This experiment confirmedthe 100 delivery of the vaccine antigen to the skin.

Proof-of-Concept In-Vivo Immunization Study:

To check the efficacy of the whole cell microparticulate vaccine, it wasadministered to 4-6 weeks old mice (Balb-C) via subcutaneous route whichserve as conventional routes of administration of vaccines. There were 3groups, subcutaneous microparticle vaccine (GnH MP), subcutaneousvaccine suspension (GnH Susp) and negative control which was receivedthe blank microparticles (n=6). There was one prime dose followed by twobooster doses 2 weeks apart. Blood samples were collected prior to primedose followed every 2 weeks. The antibody levels in the blood weremeasured using specific ELISA by plating the antigen on the plate. Theantigen specific antibody in the serum was detected using ELISA. A risein specific antibody levels is seen in FIG. 4 in groups which receivedthe vaccine when compared to the blank microparticles which serve as thenegative control after week 4.

Transdermal In-Vivo Immunization Study Using Microneedles

Since the subcutaneous route generated antibodies (Preliminary data;FIG. 8), we harness the rich immune system of the skin and deliver thevaccine via advanced microneedle patch. These microneedles are loadedwith gonorrhea microparticulate vaccine which when applied to the skin,deliver the vaccine in the epidermis and dermis layers which is rich indendritic cells and specialized Langerhans cells which will generate theimmune response. The study will be carried out in 4-6 weeks Balb-c mice.There will be following groups in the study:

1. Naïve

2. Blank Microneedles

3. Vaccine Suspension (Subcutaneous)

4. Vaccine loaded Microneedles (Transdermal)

5. Vaccine Microparticles in Microneedles (Transdermal)

Similar to previous preliminary study, there was one prime dose and twobooster doses. Blood samples will be collected prior to dosing andfollowed by every 2 weeks. The animals will be monitored for 10-12weeks. The antibody are measured as previous described using ELISA (FIG.9) which show that the groups receiving vaccine showed significantlyhigher serum IgG titers when compared to the controls—blankmicroparticles and blank microneedles after week 2. The group whichreceived the GnH vaccine microparticles in microneedles showedsignificantly higher antibody titers than the other 2 vaccine groups atweek 6 and 8 (n=6) (*p<0.001; #p<0.05). Thus we could say the potentialof transdermal route via microneedle patch as an efficient and effectivedelivery system for vaccines.

Cellular Responses in the Immunized Mice:

After the animals were sacrificed, the primary and secondary lymphoidorgans were extracted (i.e. spleen and lymph node) and made into singlecell suspensions. The single cell suspensions were stained withfluorescence-conjugated antibodies specific to T cells, helper T cells(CD4+) and cytotoxic T cells (CD8+) and quantified using flow cytometry(FIG. 10). FIGS. 10A-D show the CD4 cell counts of lymph nodes (FIG.10A) and spleens (FIG. 10B) and the CD8 cell counts in lymph nodes (FIG.10C) and spleens (FIG. 10D). The groups which received the Gonorrheavaccine microparticles incorporate in the microneedles showedsignificantly higher levels of both CD4 and CD8 T cells in immune organswhen compared to groups receiving no vaccine and blank microneedles(*P<0.05). For determination of antigen specific T cell responses,splenocytes from the various groups were plated onto a 48 well plate andre-stimulated with antigen for 16 hours and then stained withfluorescently tagged antibodies for CD4 and CD8 cells and quantifiedusing flow cytometry (FIG. 11). The groups receiving the vaccine via thesubcutaneous or transdermal route showed significantly higher cellcounts than the groups which did not receive the vaccine (*P<0.05).

EXAMPLE 1 REFERENCES

1. Ison C A, Deal C, Unemo M. Current and future treatment options forgonorrhea. Sex Transm Infect. 2013 December;89 Suppl 4:iv52-56.

2. Edwards J L, Jennings M P, Apicella M A, Seib K L. Is gonococcaldisease preventable? The importance of understanding immunity andpathogenesis in vaccine development. Crit Rev Microbiol. 2016 Jan.23;1-14.

3. Jerse A E, Deal C D. Vaccine research for gonococcal infections:where are we? Sex Transm Infect. 2013 December;89 Suppl 4:iv63-68.

4. Tapsall J W. Neisseria gonorrhoeae and emerging resistance toextended spectrum cephalosporins. Curr Opin Infect Dis. 2009February;22(l):87-91.

5. Duncan J A, Gao X, Huang M T-H, O'Connor B P, Thomas C E, WillinghamS B, et al. Neisseria gonorrhoeae Activates the Proteinase Cathepsin Bto Mediate the Signaling Activities of the NLRP3 and ASC-ContainingInflammasome. J Immunol. 2009 May 15;182(10):6460-9.

6. Zhu W, Ventevogel M S, Knilans K J, Anderson J E, Oldach L M,McKinnon K P, et al. Neisseria gonorrhoeae Suppresses DendriticCell-Induced, Antigen-Dependent CD4 T Cell Proliferation. PLOS ONE. 2012Jul. 23;7(7):e41260.

7. Liu Y, Islam E A, Jarvis G A, Gray-Owen S D, Russell M W. Neisseriagonorrhoeae selectively suppresses the development of Th1 and Th2 cells,and enhances Th17 cell responses, through TGF-β-dependent mechanisms.Mucosal Immunol. 2012 May;5(3):320-31.

8. Jerse A E, Bash M C, Russell M W. Vaccines against gonorrhea: currentstatus and future challenges. Vaccine. 2014 Mar. 20;32(14):1579-87.

9. Chablani L, Tawde S A, Akalkotkar A, D'Souza C, Selvaraj P, D'Souza MJ. Formulation and evaluation of a particulate oral breast cancervaccine. J Pharm Sci. 2012 October;101(10):3661-71.

10. Shastri P N, Kim M-C, Quan F-S, D'Souza M J, Kang S-M.Immunogenicity and protection of oral influenza vaccines formulated intomicroparticles. J Pharm Sci. 2012 October;101(10):3623-35.

11. Ubale R V, D'Souza M J, Infield D T, McCarty N A, Zughaier S M.Formulation of meningococcal capsular polysaccharide vaccine-loadedmicroparticles with robust innate immune recognition. J Microencapsul.2013;30(1):28-41.

12. Ubale R V, Gala R P, Zughaier S M, D'Souza M J. Induction of deathreceptor CD95 and co-stimulatory molecules CD80 and CD86 bymeningococcal capsular polysaccharide-loaded vaccine nanoparticles. AAPSJ. 2014 September;16(5):986-93.

13. Gao H, Pan J-C, Chen B, Xue Z-F, Li H-D. [The effect of HPV16E7 DNAvaccine transdermal delivery with microneedle array]. Zhonghua Yu FangYi Xue Za Zhi. 2008 September;42(9):663-6.

EXAMPLE 2

Preclinical Evaluation of a Novel Microneedle Based TransdermalMicroparticulate Vaccine for Metastatic Breast Cancer

Abstract

According to the National Cancer Institute, breast cancer affects one ineight women during their lives. It kills more women in USA than anyother cancer. Current treatment strategies act non-specifically againstboth tumor cells and normal cells. We have previously tested theefficacy of vaccine microparticles in a prophylactic setup and foundthat vaccination induces an immune response against cancer antigens andhelps controls tumor growth. After carrying out preliminary studiesinvolving both in-vitro and in-vivo evaluation of vaccinemicroparticles, we wanted to further investigate the therapeuticbenefits of vaccine microparticles against breast cancer in murinebreast cancer model. The immunized animals showed significantly lowertumor growth compared to the naïve animals that did not receive anytreatment. The delay in tumor growth in vaccinated animals was due to astrong immune response generated against the tumor-associated antigensencapsulated within the microparticles. We observed a significantincrease in the CD4+ T cell population. Our research strategy is tocombine chemotherapy with immunotherapy. Low dose chemotherapy has beenreported to inhibit the immunosuppressive regulatory T cells. The immuneorgans will be collected and processed to understand the immune responsegenerated after simultaneous administration of vaccine microparticlesand chemotherapeutic drug. Primary breast cancer is currently treatedwith surgical removal, chemotherapy and/or radiotherapy. But due tometastatic tumor spread many (20-25%) patients experience a relapse ofthe tumors despite these interventions. Using murine 4T1 metastaticbreast cancer cell line we have developed a microparticulate vaccineformulation. These particles were evaluated for their size, charge,surface morphology by various in vitro studies. This proposal willexplore the feasibility of eliminating residual breast cancercells/tumors and avoiding disease recurrence using a primary vaccinetreatment approach in a murine model of breast cancer.

Significance

Breast cancer (BC) is the most commonly diagnosed malignancy and is thesecond leading cause of cancer related death in American women. Thisyear over 250,000 women will be diagnosed with breast cancer and almost50,000 women will die from metastatic breast cancer. Currently there areno FDA approved vaccines for breast cancer. For this reason developmentof a therapeutic breast cancer vaccine is an area of research that needsurgent attention offering these women a better chance of a cancer freelife. Many therapeutic vaccine strategies are under clinical trials forbreast and other types of cancers (1). Most vaccines being studiedtoday, such as the gene transfer based vaccines require live culturedcells, which is time consuming and difficult to establish in manycancers. It is well known that breast cancer cells do not grow easily invitro, significantly limiting the number of patients eligible for suchclinical trials and ultimately vaccine therapy. A recent clinical trialevaluating vaccine-based therapy concluded that one of the majorproblems with gene-based cancer vaccine therapy is the delay vaccineproduction which significantly limits the access of patients to thetrial and subsequent therapy (2). Another potential problem is that thedelay in vaccine production and subsequent administration could alsoresult in a delay in treatment and progression of tumor metastasisresulting in increasing tumor burden and worsening prognosis. Thereforean optimum cancer vaccine requires a rapid production time, ease ofdelivery, and has the ability to be customizable for individualpatients. Our proposed microparticle-based vaccine approach, addressesmany of the problems associated with the current vaccine therapiesincluding the high vaccine costs. We have developed a novel formulationusing sustained release polymers encapsulating antigens in abiodegradable matrix containing immune potentiator adjuvants (3-7). Thishas been confirmed in several other studies in our laboratory withovarian (4), prostate (8) and melanoma (7) vaccines. Thus, we expect ourproposed breast cancer vaccine formulations to be very robust forinducing immunity and to overcome most of the problems associated withsoluble antigens.

We use a multi-fold approach to enhance the immunogenicity and efficacyof cancer vaccine microparticles. Following are the approaches:

Particle-based vaccination for BC: There are several challenges indeveloping an effective vaccine, which include the maintenance ofvaccine integrity and stability, avoidance of immune tolerance, andinduction of strong protective immunity. We have been studying theencapsulation of drugs in our lab as well for the past 22 years and haveshown that microparticles containing different drugs in the 0.5-2 μmsize are readily taken up by phagocytic macrophages (21). Recently, wehave made major advances in the formulation process, and have severalpatents demonstrating the production of microparticles using a modifiedspray drying methodology in a single step process (see, for example,U.S. Pat. Nos. 6,555,110, 7,105,158, and 7,425,543, the disclosures ofwhich are incorporated by reference herein in their entirety). Themicroparticles provide a depot from which the antigens a slowly releaseand cause a long lasting immune response. These microparticles protectthe antigen from being cleared out from the body thus, enhancing thevaccine stability. This will be a major advantage from the standpoint ofadvancing the vaccine formulation from bench to clinic as scale-up ofthe process can easily be achieved with no further modifications.

Adjuvant for immune-potentiation in BC: In the recent years, adjuvantslike MF59 have been incorporated in vaccine formulation to enhance thespecific immune response generated by the antigen. MF59 can potentiatethe immune response by either increasing the antibody response andinducing cell mediated immunity i.e. they have a balanced strong Th1/Th2stimulation. The use of adjuvants not only enhances immunogenicity butcould also permits the reduction in the antigen dose to be delivered invaccine thus sparing the antigen. MF59 is a squalene in water emulsionwhich is commercially been approved in Europe with more than 27 milliondoses of vaccine containing MF59 have been administered. NovartisVaccines has developed an influenza vaccine using MF59 along withinactivated, subunit seasonal prophylactic vaccine that iscommercialized successfully as Fluad® in Europe. The safety and efficacyof MF59 has been established clinically with a large database.

Temporary depletion of Treg cells: Using a low dose of cyclophosphamidethe Treg can be suppressed before vaccination. The dose ofcyclophosphamide is much lower than the chemotherapeutic dose, thusthere are no potential side effects to the patients. The Treg cellsremain depleted for a short period of 45 days and then again reachnormal levels. Thus during the vaccination regimen the Treg levels arelow and aid in generation of a strong immune response

Transdermal Vaccination:

The skin provides a unique site for the vaccination purposes as it iseasily accessible and houses various immune cells for an efficientimmune response against a range of antigens. Skin serves as a barrieragainst various pathogens and is equipped with the skin associatedlymphoid tissues (SALT) to combat any insult from invading pathogens.Various skin cells assist in generation of effective immune response.Keratinocytes are the most pre-dominant (95%) epidermal cells in theskin. They can be activated by pathogens and result in production ofcytokines, which in turn recruits dendritic cells/antigen-presentingcells to the site of action leading to initiation of the immuneresponse. Skin host's special kind of dendritic cells, the Langerhanscells. Langerhans cells comprise of only 2% of the total cell populationin the epidermis but due to their extended dendrites spread in theepideiriial layer they cover over 25% of the skin surface. These areprofessional phagocytic cells efficient in immune surveillance andfurther signaling to the T-cells present in their vicinity. Activatedmacrophages and T-cells drain into nearby lymph nodes leading to anenhanced immune response. Currently most of the vaccines areadministered via subcutaneous or intramuscular route. These have beenhighly effective in generating protective immune response but theyremain to be invasive, painful and require a skilled professional forvaccination. In an attempt to minimize some of these issues scientistshave explored the potential of delivering particulate based vaccineantigens intradermally using microneedles formulated in our laboratoryas described earlier. Microneedle arrays as the name indicates, aremicron-sized needles, which upon insertion into the skin result information of aqueous conduits forming a passage for the vaccine antigenstowards the immune-competent skin layers. Due to their short needlelength, they avoid contact with the nerve endings in the dermis thusremain to be a painless mode of immunization. This opens a new avenue ofvaccine delivery through an effective, painless and patient-friendlyroute of administration. The success of immunization via skin usingmicroneedles inspired us to evaluate the potential of delivering abreast cancer vaccine through this route. This approach further canpotentially be translated to a clinical setting where the patientundergoes a surgery for removal of the tumor and these tumor cells canserve as source of antigens for an individualized particulate vaccine,which can be administered therapeutically to avoid relapse

Specific Aims

The American Cancer society estimates approximately 232,670 women willreport for breast cancer in USA alone. According to the national cancerinstitute around 40,000 deaths are projected to occur due to breastcancer in USA (Siegel, Ma, Zou, & Jemal, 2014). Current treatmentstrategies for breast cancer involves some type of surgery to removecancerous tissue followed by chemotherapy, radiation therapy or hormonetherapy. Both chemotherapy and radiation therapy do not act specificallyagainst tumor cells and therefore have serious side effects on normalcells also due to metastatic tumor spread many (20-25%) patientsexperience a relapse of the tumors despite these interventions. Thus avaccine that can prevent the tumor growth as well as prevent metastasisof tumor cells is the need of the time.

We propose to formulate a microparticulate vaccine formulation formetastatic breast cancer by using a murine metastatic breast cancer cellline 4T1 for transdermal administration through microneedles. Wehypothesize that the microneedle based microparticulate vaccineformulation is a viable dosage form that may result in significantreduction in tumor growth as well as prevention of metastasis in murinemodel.

Aim 1: To prepare, characterize and evaluate the immunogenicity ofbiodegradable 4T1 metastatic breast cancer vaccine microparticles

Approach: Tumor associated antigens present in whole cell lysate of 4T1cell line will be added to a mix of biodegradable polymers andformulation will be spray dried to obtain the microparticles.Subsequently, the microparticles will be incorporated into microneedlesand will be characterized for physiochemical parameters and induction ofinnate immunity.

Impact: Microparticles will serve to protect the tumor associatedantigens present in the whole cell lysate vaccine formulation and resultin efficient uptake and presentation by dendritic cells.

Aim 2: To evaluate the cell surface expression on dendritic cellstreated with the microparticulate vaccine formulation.

Approach: Dendritic cells will be exposed to particulate vaccinewith/without adjuvants to determine co-stimulatory expression requiredfor activation of T and B lymphocytes

Impact: The increase or decrease in expression of surface markers on thecells will enable us to understand the interaction between the innateand adaptive immune system. Antigen presentation by dendritic cellsstimulates other immune pathways to trigger humoral and cell mediatedimmune responses.

Aim 3: To determine the efficacy of the particulate 4T1 metastaticbreast cancer vaccine administered by the microneedle based transdermalroute in murine breast cancer model

Approach: Balb/c female mice will be injected with 4T1 breast cancercells and then will be vaccinated via the transdermal route usingdissolving microneedle. Tumor volume and weight measurement will betaken regularly. At the end of the study different immune organs (e.g.lymph nodes, spleen) will be analyzed to determine if there is anyincrease in specific immune responses in treatment groups compared tocontrol groups.

Impact: A significant reduction in the tumor volume and metastasis andincrease in immune response will in treatment groups will suggest thatthe vaccine is efficacious in treating metastatic breast cancer.

Innovation

Multiple approaches will be applied to ensure potent and efficaciousbreast cancer therapy. Following innovative approaches will be combined:

Currently there is no approved FDA vaccine against breast cancer.Development of an efficacious vaccine will have a major impact in thefield of cancer immunotherapy as similar approach could be used in othertypes of cancers.

Particulate nature of vaccine ensures sustained release of antigens,higher internalization and stronger immune response than vaccinesolution

Use of vaccine adjuvants such as MF59 to enhance the immunogenicity oftumor antigens will enhance the immunogenicity of weakly immunogeniccancer antigens.

Paradigm Shift: Primary breast cancer is currently treated with surgicalresection, chemotherapy and/or radiotherapy. Despite these interventionsmany (20-25%) patients experience a relapse of the tumors due tomicrometastatic tumor spread undiagnosed at the time of surgicalresection. This proposal will explore the feasibility of eliminatingresidual breast cancer cells/tumors and avoiding disease recurrenceusing a primary vaccine treatment approach in a murine model of breastcancer. We aim to induce strong, broad and long lasting immunity, usingadjuvant such as MF 59 and alum, which have demonstrated promisingresults in vaccine efficacy studies where they act as immunepotentiator. We also aim to develop a novel treatment strategyconsisting of a simultaneous use of low dose immuno-modulatingchemotherapeutic drug and vaccine therapy for treatment of breastcancer. A low dose of cyclophosphamide, which causes not toxicity inhumans, will be used to inhibit regulatory T cell levels. We expect thiscombination to create a stronger immune response against tumor cells.With this strategy, we aim to induce strong, broad and long lastingimmunity against breast cancer resulting in a survival advantage inindividuals afflicted with this disease. Our proposal focuses on breastcancer with the goal of clinical translation. However, microparticlevaccine technology could have widespread applications in other cancersand/or infectious diseases as well.

Preliminary Studies

The goal of this study was to determine the formulation parameters of amicroparticulate vaccine for metastatic breast cancer using murine 4T1metastatic breast cancer cell line.

Specific Aim 1: To develop and characterize microparticulate formulationfor transdermal delivery of metastatic breast cancer vaccine

Vaccine Preparation

Briefly, whole cell lysate for murine breast cancer cell line (4T1) wasprepared by using hypotonic lysis buffer (10 mM Tris and 10 mM NaCl) andfurther subjected to five freeze thaw cycles at −80° C. and 37° C. for10 minutes each. At the end of last freeze thaw cycle, cell lysis wasconfirmed using trypan blue dye exclusion assay; presence of dead cellsconfirmed the end point. The whole cell lysate (WCL) thus obtained wasstored at −80° C. for further use.

Formulation Preparation

The 4T1 antigen loaded vaccine particles were prepared by spray-dryingan aqueous suspension containing whole cell lysate WCL, ethyl cellulose,cellulose acetate phthalate (CPD), hydroxyl-propyl methylcelluloseacetate succinate (HPMCAS) and trehalose using the following formuula :Whole cell lysate WCL=10% w/w, Ethyl cellulose=35% w/w, Celluloseacetate phthalate (CPD)=25% w/w, Hydroxyl-propyl methylcellulose acetatesuccinate (HPMCAS)=25% , Trehalose=5% w/w. This final mixture was thenspray dried using Buchi 290 Mini Spray Dryer (Buchi Corporation,Newcastle, Delaware) with an inlet temperature of 125° C. and outlettemperature of 80° C. The particles were stored at −20° C. until furtheruse. Adjuvant microparticles were prepared following the same procedurewith adjuvant loading of 2.5% w/w.

Vaccine and adjuvant microparticles were incorporated into dissolvingmicroneedle patches for vaccine administration. Dissolvable microneedlesmainly deploy PDMS micromolds which are made from a master structure ofmicroneedles. Briefly, polydimethylsiloxane (PDMS) was poured onto thestainless steel master structure obtained from Micropoint TechnologiesINC, Singapore. The microneedles were made using the following formula:Drug/Particles=10% w/w, Trehalose=25% w/w, Maltose=25% w/w, PVA=20% w/w,HPMC=20% w/w

The resulting suspension was then added to mold avoiding air bubbles.These molds were then centrifuged for 5-10 minutes. Then the molds weredried overnight. After overnight drying dried microneedles werecollected to use for the administration of vaccines.

Size and Zeta Potential

Spray dried particles were analyzed for their size and zeta potential.Antigen loaded microparticles were suspended in citrate buffer (100 mM,pH 4.0) and particle size was measured using Spectrex laser particlecounter (Spectrex Corp. CA). Zeta potential of these microparticles wasmeasured using Malvern Zetasizer Nano ZS (Malvern Instruments, Worcs,UK). For morphology studies, vaccine microparticles were visualizedusing scanning electron microscope (Phenom World Pure Scanning electronmicroscope). The particle size was within a range of 1-4 μm. The sizeand shape of particles was confirmed using scanning electron microscopicimages (FIGS. 12A and 12B). The particles are doughnut shaped and porousin nature. The particles have a positive zeta potential of 7 to 9 mV.Positive zeta potential helps prevent aggregation and aids uptake ofparticles by dendritic cells.

Specific Aim 2: To evaluate the immunogenicity of vaccine loadedmicroparticles.

Nitric oxide assay is an important marker for innate response.Antigen-presenting cells like dendritic cells release nitric oxide uponexposure to an antigen. In this study we found that there issignificantly higher amount of nitric oxide released in the supernatantof cells exposed to vaccine microparticles compared to vaccine solutionand blank microparticles. Vaccine microparticles induced nitric oxiderelease of 70.03+10.32 μM of nitrite compared to 10.37±4.21 μM ofnitrite by lysate solution (see FIG. 13).

Dendritic cells (DCs) are one of the major effector cells of immunesystem. They form an important part of the linkage between innate andadaptive immune response. DCs phagocytose the microparticles and lysethe microparticle in order to express the antigen on its surface(Andrianov & Payne, 1998; Hardy et al., 2013; L Thiele et al., 2001;Lars Thiele, Diederichs, Reszka, Merkle, & Walter, 2003). Therefore weevaluated the ability of dendritic cells to express the vaccine antigensas either MHC I or MHC II. Also, we evaluated the effect of delivery ofantigen via microparticles on various important cell surfaceco-stimulatory signals such as CD40 and CD80.

Dendritic cells were incubated with vaccine microparticles, blankmicroparticles and vaccine (lysate) suspension for 16 hrs. As seen inFIGS. 14A-D (induction of co-stimulatory signals—MHC I, MHC II, CD40 andCD80 on dendritic cells pulsed with 4T1 breast cancer vaccinemicroparticles), vaccine microparticles induced CD 40 and CD86expression. Both CD80 and CD40 are important for binding to T cell.There was significantly higher induction of CD 40 and CD80 in presenceof vaccine microparticles compared to vaccine solution and blankmicroparticles. The whole cell lysate contains proteins which can beexpressed as either MHC I and MHCII molecules on the antigen presentingcells. Here we can see that the antigen is presented as both MHC I andMHC II molecule. Antigen presentation is higher when given as amicroparticle compared to vaccine suspension. FIG. 15 is a graph oftumor volume measurement. FIGS. 16A-D are graphs of In vivo CD4 and CD8T cell response in different treatment groups as percent cell countmeasurement of lymph nodes CD 4 T cells (FIG. 16A), spleen CD 4 T cells(FIG. 16B), spleen CD 8 T cells (FIG. 16C) and spleen CD 8 T cells (FIG.16D). Table 2 shows a progression of metastasis to lung, lymph node andliver in different treatment groups.

TABLE 2 Lymph Groups Lung Node Liver Naïve + + + Blank MP + + − VaccineMP + − − Vaccine MP + Alum MP + MF 59 MP − − − Vaccine MP +Cyclophosphamide − − − Vaccine MP + Alum + MF 59 + − − −Cyclophosphamide Vaccine suspension + Alum + MF 59 + − + −Cyclophosphamide

EXAMPLE 2 REFERENCES

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2. Nemunaitis J, Sterman D, Jablons D, Smith J W, Fox B, Maples P, etal. Granulocyte-macrophage colony-stimulating factor gene-modifiedautologous tumor vaccines in non-small-cell lung cancer. J Natl CancerInst. 2004 Feb. 18;96(4):326-31.

3. Shastri P N, Kim M-C, Quan F-S, D'Souza M J, Kang S-M. Immunogenicityand protection of oral influenza vaccines formulated intomicroparticles. J Phaini Sci. 2012 October;101(10):3623-35.

4. Uddin A N, Bejugam N K, Gayakwad S G, Akther P, D'Souza M J. Oraldelivery of gastro-resistant microencapsulated typhoid vaccine. J DrugTarget. 2009 August;17(7):553-60.

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7. D'Souza B, Bhowmik T, Shashidharamurthy R, Oettinger C, Selvaraj P,D'Souza M. Oral microparticulate vaccine for melanoma using M-celltargeting. J Drug Target. 2012 February;20(2): 166-73.

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11. Kataoka K, McGhee J R, Kobayashi R, Fujihashi K, Shizukuishi S,Fujihashi K. Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cellsfor enhanced mucosal immunity. J Immunol Baltim Md 1950.2004 Mar.15;172(6):3612-9.

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14. Xiang R, Luo Y, Niethammer A G, Reisfeld R A. Oral DNA vaccinestarget the tumor vasculature and microenvironment and suppress tumorgrowth and metastasis. Immunol Rev. 2008 April;222:117-28.

21. Bozeman E N, Shashidharamurthy R, Paulos S A, Palaniappan R, D'SouzaM, Selvaraj P. Cancer vaccine development: designing tumor cells forgreater immunogenicity. Front Biosci Landmark Ed. 2010;15:309-20.

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34. Kubin M, Kamoun M, Trinchieri G. Interleukin 12 synergizes withB7/CD28 interaction in inducing efficient proliferation and cytokineproduction of human T cells. J Exp Med. 1994 Jul. 1;180(1):211-22.

EXAMPLE 3

Transdermal Particle Based Microneedle Against Influenza

The Influenza virus is an RNA virus, part of the Orthomyxoviridaefamily.¹ There are three main subtypes of influenza: A, B and C.¹Subtype A infects nonhuman hosts, however transmission from these hoststo humans can occur which can have a serious impact and lead to apandemic crisis.¹ Subtypes B and C only infect humans.¹

Influenza is enclosed in a lipid bilayer that houses the RNA that codesfor all the virus' proteins (FIG. 17).1 There are two majorglycoproteins, hemagglutinin (HA) and neuraminidase (NA) which arecommonly used to define the strains of all the various subtypes ofinfluenza viruses.1 These two glycoproteins undergo mutation and thusgive rise to new strains which causes a major challenge for the humanimmune system to be able to defend against all the various strains.1 Inrecent years, the matrix 2 protein (M2) which is an ion channel locatedon the membrane of the virus that is responsible for maintainingspecific conditions during viral entry into the host, has been shown tobe conserved amongst the various strains.2 This characteristic of the M2protein serves as a basis for the development of a potential universalvaccine.

According to the Center for Disease Control and Prevention (CDC), thebest method of protection against influenza is vaccination prior to thestart of influenza season.3 The major goal of vaccines is to provideimmunity, which is a term used to describe the ability of the host toprotect itself against a foreign pathogen.4 This is dependent on thecoordination of two major systems the innate and adaptive.4Microorganisms such as bacteria and viruses that infiltrate the host forthe first time are met by the innate immune system, which includemacrophages that are activated and release proteins known as cytokinesand chemokines that signal other cells to the site of infiltration.5 Avital class of immune cells that arrive to the site are dendritic cells(DCs) which help by engulfing these pathogens thus causing induction ofthe adaptive immune response.5 Following encounter with the pathogen,dendritic cells enter a developmental program referred to asmaturation.6 The function of mature DC's is not to destroy pathogens,but to become highly effective antigen-presenting cells (APCs) and carryantigens (foreign protein or molecules) to lymphoid organs and presentthem to other immune cells.5

Antigens themselves can be classified as exogenous or endogenous,exogenous implies that the antigen enters the host from the externalenvironment (i.e. injected vaccine), endogenous antigens are generatedwithin cells of the host (i.e. proteins encoded by viruses).5 As thename suggests antigen presentation is done by none other thanantigen-presenting cells (APCs) and is unique and dependent on thenature of the antigen (exogenous or endogenous).5 Exogenous antigens aredissected and fragments are displayed on molecules known as class IImajor histocompatibility complexes (MHC II) on the surface of APCs.5Endogenous antigens are dissected similarly to exogenous antigens andfragments are presented on class I major histocompatibility complexes(MHC I).5 Once antigen processing and presentation occurs, there isrequirement of cell to cell contact, also known as an immunologicalsynapse between antigen-presenting cells and a subset of immune cellsknown as T lymphocytes.5 Antigens presented on MHC class II initiatesbinding to the T cell receptor (TCR) on helper T lymphocytes categorizedas CD4+ T cells and antigen presentation on MHC class I binds to the Tcell receptor (TCR) on cytotoxic T cells categorized as CD8+ T cells.5Binding of these molecules to T cells does not necessarily result inactivation, the binding of a molecule known as intracellular adhesionmolecule 1 (ICAM-1) on APCs to leukocyte function-associated antigen 1(LFA-1) on T cells is important for T cell proliferation anddifferentiation.5 There is a phenomenon known as cross-presentationpredominantly done by mature DC's where there is transfer of endogenousantigens to the MHC class II pathway, a mechanism is which both class Iand class II pathways of antigen presentation can be used.5 The maingoal of influenza vaccination is to utilize this phenomenon ofcross-presentation to induce an enhanced and more effective immuneresponse against viral antigens of influenza.

Administration of vaccines targets the adaptive immune system which isclassified into antibody-mediated and cell-mediated machineries.4,5 Onemachinery known as antibody-mediated is produced by a group of immunecells known as B-lymphocytes.4 Upon encounter with an antigen, Blymphocytes descend into plasma cells that are responsible for secretingproteins known as antibodies that aid in destruction of the antigen.4The other machinery known as cell-mediated is regulated by Tlymphocytes.4 There are three major subsets of T lymphocytes: cytotoxic,suppression and helper.4 Cytotoxic T lymphocytes aid in the destructionof cells that may be infected and recruit other cells to destroy thepathogen that caused the infection in these cells.4 Suppressor Tlymphocytes coordinate the immune response by regulating the level ofthe response.4 Helper T lymphocytes activate other immune cells whichaid in destruction of the pathogen, i.e. plasma cells to releaseantibodies.4 Vaccines are known to activate these machineries by firstexposure of the antigen to immune cells, leading to recognition of theantigen following second exposure and thus allowing for rapid immuneresponse.4 This phenomenon is known as immunological memory.4

There are various types of antigens that can be incorporated intovaccines.6 From the emergence of vaccines, which were originallypracticed by Edward Jenner, development of influenza vaccines have beenfocused on the use of either dead, attenuated or inactivated forms ofthe virus.5 Recent discoveries in recombinant technology have led to thedevelopment of novel systems such as viral vectors, recombinantproteins, DNA and subunit vaccines.6 Virus-like particles (VLPs) haveplayed an important role in vaccine development, using recombinantmethods and the first VLP vaccine was marketed for Hepatitis B in 1986,followed by a VLP vaccine against the human papillomavirus (HPV) in2006.5 Virus-like particles (VLPs) are multiple repeats of a protein orantigen that resembles the native form and organization of the virus,minus its genome; therefore it is a safer candidate for use in avaccine.5 The nature of VLP's allow for presentation of antigenicproteins, that have the ability to enhance antibody production leadingto improved immune response.5 Up to date, VLPs have been constructed forapproximately 30 viruses including influenza.5 The hemagglutinin andneuraminidase VLPs have been constructed and used for research purposes,however the persistent challenge is that these two proteins mutate invarious strains of the influenza virus and therefore our focus is toutilize the matrix 2 protein (M2), construct a VLP and test itspotential as a vaccine.2

In order to generate a dynamic immune response, vaccines should meet twomain criteria, it should produce long-term protection and should bespecific to the antigen.8 To meet these criteria, compounds known asadjuvants are used to enhance activation of the immune system.8 In the1920's aluminum based compounds demonstrated excellent adjuvant activityand have been licensed and are widely used in human vaccines today.8Research has shown that aluminum based adjuvants enhance immunogenicityby producing a depository at the site of administration allowing forsustained release of the antigen.8 This sustained release mechanismallows for increased interface between the antigen and cells of theimmune system.8 One of the most common entry sites for infectiousdiseases including influenza is through mucosal membranes, thereforeresearchers have sought out the use of adjuvants that target mucosalmembranes to improve vaccines.9 Immune cells express a family ofproteins categorized as toll-like receptors which help in theelimination of pathogens, therefore adjuvants that target thesetoll-like receptors can enhance the immunogenicity of vaccines.10Toll-like receptor 4 (TLR-4) activation induces mucosal and systemicimmunity in influenza viral infection, through the release ofpro-inflammatory molecules.10 Monophosphoryl Lipid A (MPL-A) originallysequestered from Salmonella minnesota R595 is a TLR-4 ligand thatgenerates resistance towards viral infections by modulation of cytokinerelease.10 MPL-A is approved for human use and is incorporated incombination with aluminum hydroxide collectively referred to as ASO4 andmarketed in CervarixTM, a vaccine against the human papillomvirus(HPV).11 Similarly, this principal combination will be added to ourmatrix 2 protein virus-like particle (M2 VLP) antigen for constructionof an immunomodulating vaccine against Influenza.

Entering into delivery options for vaccines, there are several importantconsiderations and challenges for vaccine delivery.12 In order toprepare a good vaccine, there are several parameters that must becontemplated that allow for minimal change to the antigen incorporatedinto the vaccine, especially in regards to its integrity and activity.12It has been suggested in the past decade that particulate antigens havean advantage over soluble antigens in that they mimic the nature of thepathogen and are taken up better by antigen presenting cells leading toactivation of multiple immune pathways and thus a more effectiveresponse.12 Internalization of particulate antigens has also illustratedthe ability for antigen cross-presentation, suggesting another reasonwhy the immune response may be enhanced.12 Particulate antigens have aprolonged release period and can be delivered in higher doses incomparison to soluble antigens.12 There are several methods ofdelivering antigens as particulates, one of which is incorporating theminto polymers which can be tailored with specific chemical and physicalcharacteristics capable of activating pathways in the immune system.12Polymers made up of a biodegradable matrix are an attractive approachbecause they release the encapsulated entity in a controlled manner andare safe for use in development of vaccines.12 On the basis ofparticulate systems, our goal will be to encapsulate the M2 VLP into abiodegradable matrix and evaluate immune system activation.

The most widely used route for administration of vaccines has beenparenteral delivery, however a lot of focus has been placed onnon-injectable methods, one of which utilizes the skin, which is thefirst line of defense against pathogens.13 The skin is rich in antigenpresenting cells (APCs), known as Langerhans cells (LCs) in theepidermis and dermal dendritic cells in the dermis which can activatethe T and B lymphocytes and therefore is an excellent route of deliveryfor vaccines.13, 14 There are a multitude of methods that have beenutilized for delivery of proteins as well as drugs across the skin.14 Astraightforward way to deliver molecules into the skin is the use ofmicroneedles.14 This method has been illustrated to be painless anddepending on the size and length of the needles, they can permeabilizethe skin, allowing for simple delivery of biologics.14

Innovation

Current influenza vaccines on the market use whole inactivated forms ofthe virus and use multiple antigenic strains that need to be modifiedannually to produce the desired antibodies capable of protecting againstthe virus. However with the rapid rate of mutation in the two majorglycoproteins hemagglutinin (HA) and neuraminidase (NA), there is a needfor an alternative approach for development of a universal vaccineagainst influenza. Subsequently, particulate formulations thatencapsulate the matrix 2 protein virus-like particle (M2 VLP) that isconserved among all strains was designed to overcome this limitation.Moreover, the transdermal route for vaccination has been evaluated bymany research groups, employing microneedles to allow forpermeabilization of the skin allowing for delivery of therapeutics.Nevertheless, the novelty of this proposed work is underlined by thedelivery of the M2 VLP particulate vaccine into the skin to targetdermal dendritic cells to induce immunity. Additionally, to assessprotection and efficacy of the vaccine, a live virus challenge will beconducted. Exploring these objectives are intended to hopefully bring usa step closer and advance our goal in seeking a universal vaccine forinfluenza.

Preliminary Studies

Influenza is one of the most devastating infectious diseases due to theease of spread. Memory response after primary infection is critical,however due to the high rate of mutation of the virus; the antibodiesproduced in primary infection are not specific and are unable to protectagainst secondary influenza infection. The immune system is composed oftwo major classes of immunity, innate and adaptive immunity. Adaptiveimmunity is subdivided into humoral and cell-mediated responses. Thefight against influenza involves both humoral and cell-mediated immuneresponses. In addition to humoral mediated antibody production,activation of T lymphocytes, both CD4+ and CD8+ T cells are importantfor recovery against influenza infection. Priming of the host defenseusing vaccines is key for prevention of influenza infection.

Transdennal delivery of particulate vaccines has been shown to induceprotective immunity against influenza, however the mechanism of thisresponse is not well understood. It is thought that differentiation of Tcells into Th1 and Th2 cells is vital for delineation of immune cellsthat lead to immunological memory. Th1 differentiation leads to CD8+activation of cytokines that then help to convert to memory T cells. Th2differentiation leads to CD4+ activation of cytokines that can lead toboth T and B cell memory. In order to investigate this mechanism, theTh1 and Th2 immune responses were explored following transdermalvaccination with a particulate system incorporating the A/PR/8/34 (H1N1)inactivated influenza virus and delivery using a micro needle injector.

Forurulation of Microparticulate Vaccine

The particulate formulation was first prepared using the spray dryingmethod. The inactivated influenza virus was incorporated into a polymermatrix with the following composition: 20% (w/w) cellulose acetatephthalate (Aquacoat ® CPD), 35% (w/w) HPMCAS and 33% (w/w) of ethylcellulose (Aquacoat 0 ECD). Other constituents include trehalose (5%),chitosan (5%), tween 20(0.5%) and lastly Inactivated influenza virus(1.5%). Following preparation, the microparticulates were visualizedusing transmission electron microscopy (FIG. 19). Size and charge areillustrated in Table 3.

TABLE 3 Average Size 900 nm ± 7.53 Zeta Potential −12.23 mVEncapsulation Yield 86.41 ± 2.38% Percentage Yield 95.64%

Formulation of Microneedles

Mice were immunized transdermally using dissolving microneedles.Dissolving microneedles, intended for the painless transdermal releaseof encapsulated pharmaceutical agents after dermal insertion, weredeveloped as a solution to the safety issue. Dissolvable microneedlesmainly deploy PDMS micromolds which are made from a master structure ofmicroneedles (FIG. 19). Briefly, Polydimethylsiloxane (PDMS) was pouredonto the stainless steel master structure obtained from MicropointTechnologies INC, Singapore (Step 1-3; FIG. 19). The M2e VLP formulationwas then spray dried respectively. The microneedle formulation includes10% of M2e VLP, 25% trehalose, 25% maltose, 20% polyvinyl alcohol and20% hydroxypropylmethylcellulose acetate succinate (HPMCAS). Theformulation was prepared beginning with the addition of PVA, HPMC,Maltose and Trehalose to a microcentrifuge tube, followed by theaddition of ammonium hydroxide (NH4OH) to the microcentrifuge tube. Oncedissolved, this formulation was then added to a microneedle moldavoiding air bubbles (FIG. 19). These molds were then placed straightinto 50 mL centrifuge tubes. Centrifugation was done in a fixed anglecentrifuge in order to remove air bubbles and to force the formulationinto the microneedles mold. The maximum speed was 2000 rpm which wasachieved step wise, in order to avoid jerking in the rotation process,time for which centrifugation should be done was 5-10 min. After thiscentrifugation step, the backing layer was added and centrifugedrepeatedly in the same manner. The molds were placed in in tubes andthen in an incubator at 37 degrees C. overnight.

To investigate the efficacy of this M2 VLP formulation. Dendritic cellsplay a major role in immune system activation and are well known fortheir ability to release chemical messengers known as cytokines orchemokines, followed by activation of the adaptive immune system.Dendritic cells take up antigens and present them on majorhistocompatibility complexes I and II (MHC I, MHC II).

The soluble and particulate vaccines (with and without adjuvants) wereintroduced to dendritic cells with the same amount of antigen andadjuvant. The induced cells were incubated for 20 hours at 37oC with 5%CO2. Different markers can be used to evaluate the antigen presentationon APCs in ex-vivo or in-vitro studies. Some of these markers includeCD40, MHC class II, CD80, and CD86. Both expressions of CD40 and MHC IIon the surface of APCs are necessary for a successful stimulation of Tcells from APCs as demonstrated in FIGS. 20 and 21 (18). CD80 and CD86molecules are normally expressed on activated APCs and provide criticalco-stimulatory signals for T cell activation and survival (19). FIG. 20shows that M2p MP group showed statistically higher CD 40 expressioncompared to M2p solution group. Except for alum, MF59, CpG and flagellingroups, all other adjuvant treated groups showed significant increase inCD40 expression levels compared to M2P MP alone. FIG. 21 shows that theM2p MP group showed statistically higher CD 40 expression compared toM2p solution group. Except for P4 and Flag, MHC II expression waselevated in all adjuvant treated groups compared to M2p MP. Elevated MHCII expression will elicit a T-helper cell mediated response leading toantibody production against the antigen. In FIGS. 22 and 22, themicroparticulate vaccine enhanced the CD80 expression but not the CD86expression, compared to these expressions of the soluble vaccine group.In FIG. 22, the M2p MP group showed statistically higher CD80 expressionlevel compared to the M2p solution group. CD 80 expression was elevatedin Alum, CT, MF50, MPL, R848 and treated groups. In FIG. 23, there wasno significant difference in CD 86 expression between soluble andmicroparticulate M2p vaccines. CD 86 expression was elevated in R848, P4and CpG treated groups compared to M2p MP.

Table 4 shows the group, treatment, route, dose and number of doses.

TABLE 4 Dose # of Group Name Treatment Route (μg) Doses Naïve PBSTransdermal — 3 Influenza Inactivated Intramuscular 10 3 Vaccine virusFree antigen M2 VLP Transdermal 10 3 Antigen loaded M2 VLP Transdermal10 3 particulates Antigen loaded M2 VLP Transdeimal 10 3 particulatewith adjuvant

For animal experiments, 6-8 weeks old male C57BL/6 mice were immunizedwith 3 doses at 3-week intervals. Four weeks after the final boosterdose, mice were challenged with a lethal dose of mouse adaptedA/Philippines/2/82 (H3N2) live influenza virus. Mice were monitoreddaily to record changes in weight and mortality. Following 25% loss inbody weight, animals were sacrificed for further analysis. by MercerUniversity IACUC review board and conducted under the guidelines ofMercer University IACUC.

Blood samples were collected before immunization and every 3 weeks,samples were stored in −20 ° C. until analysis. Specific serum antibodyfor the matrix protein (M2) were assessed using ELISA. Coating antigenswere the M2 peptide or inactivated virus at a concentration of 200ng/well. Serum samples were used for detection of primary antibody andhorseradish peroxidase (HRP) conjugated goat anti-mouse IgG, IgG1 andIgG2a were used as secondary antibodies to determine total amount ofantibody and antibody isotypes. The substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) was used, followed by 0.3M sulfuric acid(H2504) as stop solution for detection of color. The optical density wastaken at 450 nm by a spectrophotometer. See FIG. 24.

C) Evaluation of T Cell Responses

After the animals are sacrificed, the lymph node was extracted (i.e.spleen and lymph node) and made into single cell suspensions. The singlecell suspensions were stained with fluorescence-conjugated antibodiesspecific to T cell phenotypes, helper T cells (CD4+) and cytotoxic Tcells (CD8+) and quantified using flow cytometry. See FIG. 25.

EXAMPLE 3 REFERENCES

1. Clancy S. Genetics of the influenza virus. Nat Educ. 2008;1(1):83.

2. Kim M-C, Song J-M, Eunju O, Kwon Y-M, Lee Y-J, Compans R W, et al.Virus-like particles containing multiple M2 extracellular domains conferimproved cross-protection against various subtypes of influenza virus.Mol Ther. 2013;21(2):485-92.

3. Vaccines and Immunizations Home I CDC [Internet]. [cited 2016 Jun.20]. Available from: https://www.cdc.gov/vaccines/index.html

4. Health GBD of Immunisation against infectious diseases. TheStationery Office; 2006. 486 p.

5. Janeway C A, Travers P, Walport M, Shlomchik M J. Immunobiology: theimmune system in health and disease. Vol. 1. Current Biology; 1997 6.Roldãn A, Mellado M C M, Castilho L R, Carrondo M J, Alves P M.Virus-like particles in vaccine development. Expert Rev Vaccines.2010;9(10):1149-76.

7. Soema P C, Kompier R, Amorij J-P, Kersten G F. Current and nextgeneration influenza vaccines: Formulation and production strategies.Eur J Pharm Biopharm. 2015;94:251-63.

8. Petrovsky N, Aguilar J C. Vaccine adjuvants: current state and futuretrends. Immunol Cell Biol. 2004;82(5):488-96.

9. Moldoveanu Z, Clements M L, Prince S J, Murphy B R, Mestecky J. Humanimmune responses to influenza virus vaccines administered by systemic ormucosal routes. Vaccine. 1995;13(11):1006-12.

10. Mifsud E J, Tan AC-L, Jackson D C. TLR agonists as modulators of theinnate immune response and their potential as agents against infectiousdisease. Front Immunol. 2014;5:79.

11. Kim K S, Park S, Ko K-N, Yi S, Cho Y J. Current status of humanpapillomavirus vaccines. Clin Exp Vaccine Res. 2014;3(2):168-75.

12. Xu Q, Hashimoto M, Dang T T, Hoare T, Kohane D S, Whitesides G M, etal. Preparation of monodisperse biodegradable polymer microparticlesusing a microfluidic flow-focusing device for controlled drug delivery.Small. 2009;5(13):1575-81.

13. Song J-M, Wang B-Z, Park K-M, Van Rooijen N, Quan F-S, Kim M-C, etal. Influenza virus-like particles containing M2 induce broadly crossprotective immunity. PloS One. 2011;6(1):e14538.

14. Kim K K, Pack D W. Microspheres for drug delivery. In: BioMEMS andBiomedical Nanotechnology. Springer; 2006 p. 19-50.

15. Savina A, Amigorena S. Phagocytosis and antigen presentation indendritic cells. Immunol Rev. 2007;219(1):143-56.

16. Théry C, Amigorena S. The cell biology of antigen presentation indendritic cells. Curr Opin Immunol. 2001;13(1):45-51.

17. Germain R N, Margulies D H. The biochemistry and cell biology ofantigen processing and presentation. Annu Rev Immunol.1993;11(1):403-50.

18. Burgdorf S, Kurts C. Endocytosis mechanisms and the cell biology ofantigen presentation. Curr Opin Immunol. 2008;20(1):89-95.

19. Trombetta E S, Mellman I. Cell biology of antigen processing invitro and in vivo. Annu Rev Immunol. 2005;23:975-1028.

20. O'Hagan D T, Singh M, Ulmer J B. Microparticle-based technologiesfor vaccines. Methods San Diego Calif. 2006 September;40(1):10-9.

21. Prausnitz M R, Langer R. Transdermal drug delivery. Nat Biotechnol.2008;26(11):1261-8.

22. Moran T M, Park H, Fernandez-Sesma A, Schulman J L. Th2 responses toinactivated influenza virus can be converted to Th1 responses andfacilitate recovery from heterosubtypic virus infection. J Infect Dis.1999;180(3):579-85.

23. Avetisyan G, Ragnavolgyi E, Toth G T, Hassan M, Ljungman P.Cell-mediated immune responses to influenza vaccination in healthyvolunteers and allogeneic stem cell transplant recipients. Bone MarrowTransplant. 2005;36(5):411-5.

24. Wang B-Z, Gill H S, Kang S-M, Wang L, Wang Y-C, Vassilieva E V, etal. Enhanced influenza virus-like particle vaccines containing theextracellular domain of matrix protein 2 and a Toll-like receptorligand. Clin Vaccine Immunol. 2012;19(8):1119-25.

25. Kang S-M, Yoo D-G, Lipatov A S, Song J-M, Davis C T, Quan F-S, etal. Induction of long-temi protective immune responses by influenza H5N1virus-like particles. PLoS One. 2009;4(3):e4667.

26. Kim T S, Sun J, Braciale T J. T cell responses during influenzainfection: getting and keeping control. Trends Immunol.2011;32(5):225-31.

27. Bramwell V W, Perrie Y. Particulate delivery systems for vaccines:what can we expect? J Pharm Pharmacol. 2006;58(6):717-28.

EXAMPLE 4

Transdermal Particle Based Microneedle Against Human Papilloma Virus(HPV)

Human papillomaviruses (HPVs) are transmitted through sexual contact andmost people are infected with HPV shortly after the onset of sexualactivity (World Health Organization, 2015). There are more than 100different types of HPVs, which are classified into two categories:non-oncologic and oncologic. Patients infected with the non-oncologicHPVs experience body warts on hands and feet, whereas these infectedwith the oncologic HPVs face development of genital warts orcarcinogenic symptoms. More than 40 various HPV serotypes cause 90%genital warts such as HPV6, 11, 31, 33,45, 52 and 58. Two HPV serotypes(16 and 18) are responsible for approximately 70% of cervical cancersand precancerous cervical lesions (Centers for Disease Control andPrevention, 2015, p. 1). Several symptoms of cervical cancer tend toappear only after the cancer has reached an advanced stage, whichinclude irregular, intermenstrual or abnormal vaginal bleeding aftersexual intercourse; back, leg or pelvic pain; fatigue, weight loss andloss of appetite. It takes 15 to 20 years for cervical cancer to developin women with a normal immune system, while 5 to 10 years in women witha weakened immune system, such as those with untreated HPV infection.

Two commercial vaccines (Gardasil® from Merk, and Cervarix® fromGlaxoSmithKline) are widely available in North America and Europe. TheCenters of Disease Control and Prevention (CDC) recommends boys andgirls to get vaccinated against HPV, especially between ages of 9 to 26.Both vaccines consist of HPV16 and HPV18 to prevent cervical cancers.Additionally, Gardasil® contained 9 different HPV serotypes (HPV 6, 11,16, 28, 31, 33, 45, 52, and 58) which enhance the protection fromcancers (cervical, vulvar, vaginal and anal) and genital warts (Centersfor Disease Control and Prevention, 2015). However, the high cost ofvaccine and trained personnel are a significant financial burden,especially in developing countries. Moreover, the vaccine needscold-chain storage that adds to the vaccine cost. To overcome thechallenges of injectable vaccines, vaccine solutions can be convertedinto micro/nano particles using a spray-drying or lyophilization process(Prathap Nagaraja Shastri et al., 2015).

The spray drying process first evolved several decades ago, with thesudden need to reduce the transport weight of food and other materials(R. P. Patel, M. P. Patel, & A. M. Suthar, 2009). Nowadays, spray dryingprocess is extensively employed in the pharmaceutical field because ofseveral advantages: (i) single step processing, (ii) easy to scale-up,and (iii) continuous processing operation. The functional principle ofthe spray drying process is based on the atomization of a liquid feedinto very small droplets within a hot drying gas leading to flash dryingof the droplets into solid particles (Ano et al., 2011). The particlesare then separated from the drying gas, using a cyclone and/or a filterbag, which yields a final spray dried product (Filipe Gaspar, 2014).

Vaccine nano/microparticles consist of a single or multiple antigensincorporated into a polymer matrix along with targeting ligands,adjuvants, and cytokines (shown as FIG. 26). Antigens can be inactivatedwhole cells (virus, bacteria), a part of the cells (capsid proteins,DNAs, peptides, polysaccharides) or toxoids. For examples, virus-likeparticles (VLPs) are made of capsid protein L1 and L2 of HPVs and areused as antigens in Gardasil® and Cervarix. Virus-like-particles (VLPs)are genetically engineered particles similar in size and structure tothe virus but do not possess viral genomes, hence they lack the abilityto replicate and cause infection in the host.

Polymer matrices such as polylactic-co-glycolic acid) (PLGA),hydroxypropyl methylcellulose (HPMC), hypromellose acetate succinate(HPMCAS), bovine serum albumin (BSA), cellulose acetate phthalate (CPD)and cyclodextrins (CDs) have been investigated as potential polymers.Selected polymers should be biodegradable and biocompatible in humans,to prevent any toxicity concerns.

Encapsulated antigens are portrayed as pathogens or foreign substancesand are therefore taken up better by antigen presenting cells (APCs) andactivate the innate and adaptive immune system.

Micro/nanoparticulate delivery systems that contain the antigen within apolymeric matrix, aid in the delivery of antigen/adjuvant for aprolonged period of time. Moreover, antigens delivered by a particulatecarrier can enhance uptake by immune cells owing to their size, surfacecharge and morphology. Several publications from our lab have proved theefficacy of using microparticles for delivering cancer and infectiousdisease antigens.

Adjuvants have been studied and employed in vaccines for decades inorder to improve, expedite, and prolong specific immune responsesproduced by vaccine antigens including increase in antibody responses,induction of cell mediated immunity, and reduction in dose of antigenand the number of doses required for vaccination (13). For a successfulimmune response to a vaccine, there are four classes of signals: (a)antigen, (b) co-stimulation of immune cells including antigen-presentingcells (APCs), (c) immune system modulation, and (d) activation of innateimmune response (14) (15). Various adjuvants utilize their distincteffects via different mechanisms to stimulate the immune system, andhence it is essential to appoint appropriate adjuvants for a specificgiven antigen. Adjuvants can be classified into two types: deliverysystem and immune potentiator (16). Some adjuvants function as antigendelivery systems such as alum, calcium phosphate tyrosine liposomes,virosomes, emulsions micro/nano particles (MF59, ISCOMS), and virus-likeparticles, because these particulate adjuvants increase antigenstability and allow them to be presented for an extended period of time(prolonging the signal of the antigen) (17). Delivery system basedadjuvants are often taken up by phagocytosis into antigen presentingcells (APCs), and they can also induce an immune response, signaling andindirectly activating APCs. Immune potentiators are purified componentsof bacterial cells or viruses; thus, they are recognized as “dangersignals” by receptors present on immune cells (APCs) (15). Immunepotentiators directly stimulate all the necessary signals for an immuneresponse to an antigen. A major category of immune potentiators istoll-like receptor (TLR) agonists, which activate signaling pathways totrigger innate immune responses. Some examples of adjuvants that act asTLR agonists include MPL and synthetic derivatives, muramyl dipeptideand derivatives, CpG oligonucleotides, alternative bacterial or viralcomponents (flagellin), saponins, dsRNA, and resiquimod (16). Sincedelivery system based adjuvants elevate the amount of antigen that reachAPCs and immune potentiators mainly activate these APCs; combinations ofadjuvants from both classes can be used to maximize potency of avaccine.

Furthermore, in our study we have used adjuvants, along with the antigenwhich when used in combination enhance their ability to produce a robustimmune response. We propose that adjuvants when combined with themicroparticulate vaccine will potentiate the immune response and resultin improved efficacy by generation of an antigen-specific antibodyresponse. Moreover, adjuvants enable the vaccine to produce long-termimmunity in case of re-exposure to virus.

Innovation

Conventionally, administration of influenza vaccines has utilized theintramuscular route, because this route provides long-term efficacy andsafety. However, the use of needles is still somewhat feared by society.A lot of effort and time has been spent in developing alternative routesfor administration of vaccines and medications. Recent discoveries haveshown the skin to be an excellent source of immune cells and can be usedas a strategy for vaccine administration. The transdermal route has beenwidely accepted due to its, non-invasive, easy-to-use, needle-freestrategy and especially because of the immunocompetency of the skin. Intransdermal vaccination, dendritic cells and Langerhans cells (LCs) thatreside in the dermal layer of the skin have the ability to capture theantigen, migrate to the secondary lymphoid organs and present theantigen to the T cells to generate adaptive immune responses (12).

Hence, we hypothesize that transdermal delivery of the vaccine byencapsulating a viral antigen such as the HPV 16 VLP in a biodegradablematrix may not only result in better stability and uptake by Langerhanscells/ dendritic cells but will also provide enhanced antigenpresentation and recognition by the immune system. The innovation in ourcurrent approach lies in our proposed strategy, which is to develop,characterize and assess a micro/nanoparticulate vaccine againstInfluenza. Moreover, we have evaluated the interaction between theinnate and adaptive immune systems by measuring co-stimulatoryexpression on dendritic cells exposed to the particulate vaccinewith/without adjuvants. This in vitro preliminary data has prompted usto test the immune efficacy of the vaccine when delivered by thetransdermal route. We hypothesize that our particulate vaccine willinduce specific antibodies to neutralize the human papilloma virus andcytotoxic T lymphocytes, which will eliminate the virus, infected cells.Since our particles exist in the dry powder form, they are thereforestable under normal refrigeration conditions.

Preliminary Studies

Formulation and Characterization of HPV VLP Microparticles

The goal of this study was to determine the formulation parameters of amicroparticulate vaccine for Influenza using the HPV 16 VLP. The HPV VLPwas incorporated into an enteric-coated polymer matrix in thisformulation. This matrix consisted of celluloseacetatephthalate (CPD)hydroxypropylmethylcellulose acetate succinate (HPMCAS), ethylcellulose(EC), trehalose and glycol chitosan polymers First, CPD dispersion (30%w/v) was diluted in deionized water with a concentration of five mg/mlunder stirring. CPD and HPMCAS were dissolved separately using 1N sodiumhydroxide to make final solutions at pH of 6.0 and 8.0, respectively.The mixture of CPD, HPMCAS, and EC was obtained as mentioned above, andthe final solution pH was adjusted to be 7.0. Glycol chitosan was thenadded along with HPV 16 VLP and trehalose. In addition, Tween-20 wasadded to enhance the smooth surface of the microparticles (MPs). Thesolution was stirred at 50 rpm during the spraying process using a BuchiB290 spray dryer to maintain its homogeneity. Microparticulate adjuvantswere formulated using the same procedure as the vaccine microparticles.

The microparticles (See FIG. 27) were characterized and analyzed fortheir morphology using the Phenom Pure Desktop® scanning electronmicroscopy (SEM). The average percent yield of the MPs was 90%+5.3%(w/w) after the spray drying process. The size of MPs ranged from 1.0 μmto 5.5 μm with an average size of 3.76+0.84 μm.

Evaluation of Innate Immune Response Using Murine Dendritic Cells

Sodium thioglycolate was injected into the intraperitoneal cavity ofSwiss Webster (6-8 week old) to recruit the APCs (19) (20). After fourdays, these mice were euthanized and PBS was injected intraperitoneallyinto each mouse to harvest cells from the cavity. Collected cells werewashed with twice. After these APCs adhered to the bottom of a flask andthe supernatant was removed and complete RPMI media was added in thepresence of ConA. The harvested APCs (5×104 cells/well) were seeded inDulbecco's complete media in a 96 well plate. The soluble andparticulate vaccines (with and without adjuvants) were introduced to theAPCs with the same amount of antigen and adjuvant. The induced APCs wereincubated for 20 hours at 37° C. with 5% CO2. Supernatants werecollected and the amount of nitric oxide released in each group wasanalyzed using the Greiss chemical method. The plate was read at awavelength of 540 nm (21). The nitric oxide assay was performed toinvestigate the recognition of vaccine and adjuvants by the APCs (22).Nitric oxide (NO) is released in the process of conversion of arginineto citrulline by nitric oxide synthase during the recognition andphagocytosis of antigen from APCs (23). A higher level of NO releasedindicates a stronger activation of APCs by the vaccines. FIG. 28 is agraph of nitric oxide concentration of various solutions.

Assessment of Adaptive Immune Response by Observing the Cell SurfaceExpression on Dendritic Cells Exposed to the Microparticulate VaccineFormulation with Adjuvants

Similarly to the innate immunity, after stimulating the antigen withAPCs for 20 hours, the supernatant was removed to perform the nitricoxide assay. These cells were gently washed with PBS. Trypsin-EDTAsolution was used to collect the APCs and Hank's Balanced Salt Solution(HBSS) was used to wash prior to an incubation with antimouse-CD80 PE,antimouse-CD86 FITC, antimouse-CD40 PE, antimouse-MHC II (FITC). After1-hour incubation, the cells were washed twice with HBSS, then analyzedfor the specific cell surfaced markers using BD Accuri® C6 flowcytometer.

Different markers can be used to evaluate the antigen presentation onAPCs in ex-vivo or in-vitro studies. Some of these markers include CD40,MHC class II, CD80, and CD86. Both expressions of CD40 and MHC II on thesurface of APCs are necessary for a successful stimulation of T cellsfrom APCs as demonstrated in FIGS. 29 and 30 (18). In this study, theparticulate vaccine enhanced both MHC II and CD40 expressions (p<0.001),compared to the soluble vaccine. Except P4 and flagellin, all otheradjuvants enhanced highly MHC II expression (p<0.001) on the cellsurface, compared to the M2p MP group. The highest CD40 expressions werewas found in the presence of R848 and P4 (p<0.001), followed by CT andMPL (p<0.01). Flagellin did not enhance either CD40 or MHC IIexpression.

CD80 and CD86 molecules are normally expressed on activated APCs andprovide critical co-stimulatory signals for T cell activation andsurvival (19). In FIGS. 31 and 32, the microparticulate vaccine enhancedthe CD80 expression but not the CD86 expression, compared to theseexpressions of the soluble vaccine group. Except CpG and flagellin, allother adjuvants induced statistically (p<0.001) the CD80 expression.R848 and CpG enhanced the highest (p<0.001) expression of CD86, followedby alum and P4 treated groups, compared to the vaccine MP treated group.

The human papilloma virus type 16 virus-like particles (HPV 16 VLP) wasprovided by the Center for Disease Control and Prevention (CDC). The HPV16 VLP was incorporated into a cellulose polymer mix of celluloseacetate phthalate (Aquacoat® CPD), HPMCAS and ethyl cellulose (Aquacoat®ECD). Other constituents included trehalose, chitosan, tween 20 and,lastly, the HPV 16 VLP. This suspension was spray dried using the B-290spray dryer to obtain particulates.

Mice were immunized transdermally using dissolving microneedles.Dissolving microneedles, intended for the painless transdermal releaseof encapsulated pharmaceutical agents after dermal insertion, weredeveloped as a solution to the safety issue. Dissolvable microneedlesmainly deploy PDMS micromolds which are made from a master structure ofmicroneedles as illustrated below. Briefly, Polydimethylsiloxane (PDMS)was poured onto the stainless steel master structure obtained fromMicropoint Technologies INC, Singapore. The HPV 16 VLP formulation wasthen spray dried respectively. The microneedle formulation included 10%of M2e VLP, 25% trehalose, 25% maltose, 20% polyvinyl alcohol and 20%hydroxypropylmethylcellulose (HPMC). The formulation was preparedbeginning with the addition of PVA, HPMC, Maltose and Trehalose to amicrocentrifuge tube, followed by the addition of Ammonium hydroxide(NH4OH) to the microcentrifuge tube. Once dissolved, this formulationwas then added to a microneedle mold avoiding air bubbles. These moldswere then placed straight into 50 mL centrifuge tubes. Centrifugationwas done in a fixed angle centrifuge in order to remove air bubbles andto force the formulation into the microneedles mold. The maximum speedwas 2000 rpm, which was achieved step wise in order to avoid jerking inthe rotation process, time for which centrifugation should be done was5-10 min. After this centrifugation step, the backing layer was addedand centrifuged repeatedly in the same manner. The molds were placed intubes and then in an incubator at 37 degrees C. overnight.

For animal experiments, 4-6 week old female Balb/c mice were immunized,with 3 doses, 1 prime dose and 2 booster doses at 4-week intervals. Fordetermination of long-term efficacy of the vaccine, the mice antibodyresponses were monitored for a 40-week period, followed by which theywere sacrificed for further analysis. The groups for the study are shownin Table 5.

TABLE 5 Treatment groups receiving HPV 16 VLP Vaccines Group NameTreatment Route Dose (μg) # of Doses Solution HPV 16 VLP Transdermal 20(Prime) 3 Vaccine 10 (Booster) Microparticulate HPV 16 VLP Transdermal20 (Prime) 3 Vaccine 10 (Booster)

All animal experiments were approved by Mercer University IACUC reviewboard and conducted under the guidelines of Mercer University IACUC.

Blood samples were collected before immunization and beginning at week5. Samples were stored in −20 ° C. until analysis. Specific serumantibody for HPV 16 was assessed using ELISA. Plates were coated with100μg/well of HPV VLPs. Serum samples were used for detection of primaryantibody and alkaline phosphatase (AP) conjugated goat anti-mouse IgGwas used as secondary antibody to determine total amount of antibody.The alkaline phosphatase substrate was used for detection of color. Theoptical density was taken at 405 nm on the Biotek Synergy ELISA platereader.

After the animals are sacrificed, the primary and secondary lymphoidorgans were extracted (i.e. spleen and lymph node) and made into singlecell suspensions. The single cell suspensions were stained withfluorescence conjugated antibodies specific to T cell phenotypes, helperT cells (CD4+) and cytotoxic T cells (CD8+) and memory B and T cellphenotypes, memory T cells (CD45R and CD62L) and memory B cell (CD27)quantified using flow cytometry.

EXAMPLE 4 REFERENCES

1. Centers for Disease Control and Prevention. Seasonal Influenza (Flu)[Internet]. 2014. Available from:http://www.cdc.gov/flu/about/disease/index.htm

2. Akalkotkar, A., Tawde, S. A., Chablani, L., & D'Souza, M. J. (2012).Oral delivery of particulate prostate cancer vaccine: in vitro and invivo evaluation. Journal of Drug Targeting, 20(4), 338-346.http://doi.org/10.3109/1061186X.2011.654122

3. Bhowmik, T., D'Souza, B., Shashidharamurthy, R., Oettinger, C.,Selvaraj, P., & D'Souza, M. J. (2011). A novel microparticulate vaccinefor melanoma cancer using transdermal delivery. Journal ofMicroencapsulation, 28(4), 294-300.http://doi.org/10.3109/02652048.2011.559287

4. Chablani, L., Tawde, S. A., Akalkotkar, A., D'Souza, C., Selvaraj,P., & D'Souza, M. J. (2012). Formulation and evaluation of a particulateoral breast cancer vaccine. Journal of Pharmaceutical Sciences, 101(10),3661-3671. http://doi.org/10.1002/jps.23275

5. Shastri, P. N., Kim, M.-C., Quan, F.-S., D'Souza, M. J., & Kang,S.-M. (2012). Immunogenicity and protection of oral influenza vaccinesformulated into microparticles. Journal of Pharmaceutical Sciences,101(10), 3623-3635. http://doi.org/10.1002/jps.23220

6. Li, N., Peng, L.-H., Chen, X., Nakagawa, S., & Gao, J.-Q. (2011).Transcutaneous vaccines: novel advances in technology and delivery forovercoming the barriers. Vaccine, 29(37), 6179-6190.http://doi.org/10.1016/j.vaccine.2011.06.086

7. Smith D M, Simon J K, Baker J R. Applications of nanotechnology forimmunology. Nat Rev Immunol. 2013 Aug;13(8):592-605.

8. Maisonneuve C, Bertholet S, Philpott D J, De Gregorio E. Unleashingthe potential of NOD- and Toll-like agonists as vaccine adjuvants. ProcNatl Acad Sci USA. 2014 Aug. 26;111(34):12294-9.

9. Derek T O'Hagan. New Generation Vaccine Adjuvants [Internet].Novartis Vaccines and Diagnostics; Available from:http://www.roitt.com/elspdf/Newgen_Vaccines.pdf

10. Mohan T, Verma P, Rao D N. Novel adjuvants & delivery vehicles forvaccines development: a road ahead. Indian J Med Res. 2013November;138(5):779-95.

11. Gupta R K, Siber G R. Adjuvants for human vaccines--current status,problems and future prospects. Vaccine. 1995 October;13(14):1263-76.

12. Batista-Duharte A, Lindblad E B, Oviedo-Orta E. Progress inunderstanding adjuvant immunotoxicity mechanisms. Toxicol Lett. 2011Jun. 10;203(2):97-105.

13. Cohn Z A. Activation of mononuclear phagocytes: fact, fancy, andfuture. J Immunol Baltim Md 1950. 1978 September;121(3):813-6.

14. Lagasse E, Weissman I L. Flow cytometric identification of murineneutrophils and monocytes. J Immunol Methods. 1996 Oct.16;197(1-2):139-50.

15. Zughaier S M, Tzeng Y-L, Zimmer SM, Datta A, Carlson R W, Stephens DS. Neisseria meningitidis lipooligosaccharide structure-dependentactivation of the macrophage CD14/Toll-like receptor 4 pathway. InfectImmun. 2004 January;72(1):371-80.

16. Roper R L. Antigen presentation assays to investigateuncharacterized immunoregulatory genes. Methods Mol Biol Clifton N.J.2012;890:259-71.

17. Kohchi C, Inagawa H, Nishizawa T, Soma G-I. ROS and innate immunity.Anticancer Res. 2009 March;29(3):817-21.

EXAMPLE 5

Formulation and Testing of a Microneedle-Based Particulate Vaccine ForRsv

BACKGROUND

Respiratory Syncytial Virus (RSV) is the leading cause of bronchiolitisin infants and immunocompromised adults. It is estimated thatapproximately 3.4 million children are annually hospitalized due toRSV-related illnesses and 160,000 people die from RSV infectionworldwide (Nair et al., 2010). The past few decades have been spent indeveloping a promising strategy to combat the virus either using subunitvaccines, attenuated viruses or live vector vaccines. With thecentralized controversy surrounding the disease i.e., the tragic outcomeof vaccinated children who developed vaccine-enhanced respiratorydisease, in the 1960s with alum-adjuvanted, formalin inactivated RSV;there still remains a large barrier before the licensure of an RSVvaccine (H. W. Kim et al., 1969). RSV has 10 genes that encode 11proteins. Among them are the F, fusion protein and G, glycoprotein whichare important antigenic proteins expressed on the surface of the virusand a target for neutralizing antibodies that facilitate a protectiveimmune response in the patient (Murawski et al., 2010).

There are multiple subunit licensed vaccines that use specific proteinsthat have similarities to the native fomi of the virus, one of which areVLPs. Virus-like-particles (VLPs) are genetically engineered particlessimilar in size and structure to the virus but do not possess viralgenomes, hence they lack the ability to replicate and cause infection inthe host. Recombinant baculovirus-expressed VLPs containing RSV-F and/orG glycoproteins, were shown to stimulate antigen-specific antibodyresponses and defend against RSV infection in murine models (K.-H. Kimet al., 2015; Murawski et al., 2010; Quan et al., 2011). Encapsulatedantigens are portrayed as pathogens or foreign substances and aretherefore taken up better by antigen presenting cells (APCs) andactivate the innate and adaptive immune system. Micro/nanoparticulatedelivery systems that contain the antigen within a polymeric matrix, aidin the delivery of antigen/adjuvant for a prolonged period of time.Several publications from our lab have proved the efficacy of usingmicroparticles for delivering cancer and infectious disease antigensthrough the oral, transdermal and subcutaneous routes. (Akalkotkar,Tawde, Chablani, & D'Souza, 2012; Bhowmik et al., 2011; Chablani et al.,2012; Shastri, Kim, Quan, D'Souza, & Kang, 2012). Our approach is toincorporate RSV Fusion protein VLPs in a mix of biodegradable polymersand spray dry the formulation to obtain microparticles.

Conventionally, administration of vaccines has utilized theintramuscular route, because this route provides long Willi efficacy andsafety. However, the use of needles is still somewhat feared by society.A lot of effort and time has been spent in developing alternative routesfor administration of vaccines and medications. Recent discoveries haveshown the skin to be an excellent source of immune cells and can be usedas a strategy for vaccine administration. The transdermal route has beenwidely accepted due to its, non-invasive, easy-to-use, needle-freestrategy and especially because of the immunocompetency of the skin. Intransdermal vaccination, dendritic cells and Langerhans cells (LCs) thatreside in the dennal layer of the skin have the ability to capture theantigen, migrate to the secondary lymphoid organs and present theantigen to the T cells to generate adaptive immune responses (Li, Peng,Chen, Nakagawa, & Gao, 2011). Hence, we hypothesize that transdermaldelivery of the vaccine by encapsulating a viral antigen such as thefusion protein from the surface of the RSV virus in a biodegradablematrix may not only result in better uptake but will also provideenhanced antigen presentation and recognition by the immune system.

Adjuvants are crucial compounds used in combination with vaccineantigens to enhance their ability to produce a stronger immune response.They are minimally toxic and have no long lasting immune effects whengiven alone. Specific aims 1 and 2 have used adjuvants with vaccine topotentiate the immune response against the disease. Alum, an adjuvantdelivery system has been widely used in human vaccines for decades. Thehypothesized mechanisms of Alum include enhanced antigen uptake by APCs,improved MHC II expression and antigen presentation (Dubensky & Reed,2010). Monophosphoryl lipid A (MPL®) is a Toll-like receptor-4 agonistthat induces a strong cellular (T cell mediated) immune response.Another approved adjuvant, MF59™ is a squalene in water nano-emulsionthat shows cell-mediated/antibody responses and results in secretion ofcytokines and chemokines by DCs and macrophages. Pneumococcal surfaceadhesion A-derived peptide (P4) has been recently explored as anadjuvant since it has shown enhanced opsonophagocytosis in some studies(Rajam, Anderton, Carlone, Sampson, & Ades, 2008). R848 is animidazoquinoline compound that activates immune cells via the TLR7/8pathway.

Innovation

Currently there is no safe and efficacious treatment regimen to treatinfections caused by RSV. Drugs such as corticosteroids, antibiotics andbronchodilators are primarily administered to alleviate the symptoms incomplicated cases. Ribavirin, an antiviral drug has been approved fortreatment of RSV infections. In high-risk infants a novel approvedmonoclonal antibody; palivizumab (Synagis®) is approved to prevent severlower respiratory tract infections and has markedly reduced risk ofhospitalization. However, the high cost of therapy along with no longterm memory response to protect against future RSV infections,necessitates a need for a safe and potent vaccine.

The innovation in our current approach lies in our proposed strategywhich is to develop, characterize and assess a micro/nanoparticulatevaccine against RSV, which has no licensed vaccine till date. Moreover,we have evaluated the interaction between the innate and adaptive immunesystems by measuring co-stimulatory expression on dendritic cellsexposed to the particulate vaccine with/without adjuvants. This in vitropreliminary data has prompted us to test the immune efficacy of thevaccine when delivered by the transdermal route.

We hypothesize that our particulate vaccine will induce specificantibodies to neutralize RSV and cytotoxic T lymphocytes which willeliminate the virus infected cells. Since our particles exist in the drypowder form, they are therefore stable under normal refrigerationconditions (recent unpublished data).

Preliminary Studies

Formulation and Characterization of RSV-F VLP Microparticles

The goal of this study was to determine the formulation parameters of amicroparticulate vaccine for RSV using F Fusion protein virus-likeparticles (VLPs). A unique blend of cellulose polymers and chitosan wasutilized to formulate microparticles. Briefly 0.5% (w/w) F-VLP solutionwas incorporated into a mixture of cellulose acetate phthalate,hydroxypropylmethylcellulose acetate succinate (HPMCAS), ethylcellulose,trehalose and chitosan polymers. The aqueous suspension was subsequentlyspray-dried using the Buchi-290 spray dryer to obtainmicro/nanoparticles. The microparticles were characterized and analyzedfor their size, surface charge, encapsulation efficiency and antigenstability. The Malvern Zetasizer® Nano ZS was used to carry out size andsurface potential measurements. Particles were suspended in citric acid(10 mM, pH 3.8) for 10 minutes and centrifuged. The particles wereresuspended in deionized water and later analyzed by the instrument. Themicroparticle images were captured on carbon sheets and observed under20 kV at 7500× using the scanning electron microscope (See FIG. 34).

Table 6 summarizes the physicochemical characteristics of the spraydried microparticles.

TABLE 6 Characterization of Vaccine Microparticles Product Yield 88-93%Particle Size 1-2 μm Surface Charge 25 ± 2 mV Protein Content 80-85%

To evaluate antigen integrity, SDS PAGE was performed for the VLPmicroparticulate formulation as seen in FIG. 35. The spray driedmicroparticles were added to PBS, vortexed for 10 minutes followed byincubation at 37° C. for 10 minutes to extract the antigen from thematrix to check antigen integrity and encapsulation efficiency. Tenmicrograms equivalent of F-VLP was loaded onto the acrylamide gel.Precision protein plus standards were run as a control. We found thatthe F-VLP remained intact after spray drying when compared to the F-VLPsuspension lane.

Evaluate Immunogenicity of Vaccine Microparticles Using Murine DendriticCells

After characterization, we tested the in vitro immunogenicity of the RSVF-VLP microparticles using a dendritic cell line, DC 2.4. Dendriticcells are responsible for pathogen recognition and eradication byreleasing cytokines such as TNF-α, nitric oxide (NO) and IFN-γ. Theincrease in levels of nitrite may be related to enhanced antigenrecognition and delivery to dendritic cells. NO is also known toeradicate viruses by nitrosation of cysteine residues within keyproteins required for replication purposes (Wink et al., 2011).

The microparticles' ability to generate an innate immune response wasevaluated using the nitric oxide assay. Cells were plated in 24 wellplates following which blank MPs, RSV F-VLP suspension and vaccine MPswere added and incubated with the cells for 20 hrs. The VLP-MPs werecompared with controls of blank MPs and VLP solution. Subsequently, thecell supernatant was analyzed for nitric oxide (NO) levels (Ubale,D'Souza, Infield, McCarty, & Zughaier, 2013) using the Griess Test. Theresults proved that the F-VLP microparticles were immunogenic, byactivating the innate immune system. FIG. 36 shows the amount of nitricoxide released (μM) from DC 2.4 cells when exposed to Cells Only, BlankMP, RSV F-VLP Suspension, RSV F-VLP MP (*p<0.05). There was asignificant release of nitric oxide seen in supernatant of cellsreceiving RSV F-VLP MPs. FIG. 37 shows Amount of nitric oxide releasedfrom DC 2.4 cells when exposed to VLP Suspension, RSV VLP MP and RSV VLPMP+Alum, MPL A and MF59 (*p<0.05) There was a significant release ofnitric oxide seen in supernatant of cells receiving RSV F-VLP+Alum/ MF59MPs.

Since the RSV F-VLP microparticles demonstrated immunogenicity, we wereinterested to understand the interaction between the innate and adaptiveimmune responses. Vaccines are required to be efficiently taken up andprocessed by dendritic cells and presented on major histocompatibilitycomplex I or II molecules along with co-stimulatory molecule expressionsuch as CD40 and CD80/86 that are required for T and B cell activationand survival, which are critical for an adaptive and hence a memoryresponse after vaccination. Following the nitric oxide assay we furtherexamined the surface co-stimulatory expression on dendritic cellstreated with RSV F-VLP microparticles alone and with adjuvants, whichare substances that enhance the antigen specific response. Briefly, thecells were washed with PBS and detached using Trypsin EDTA. Each groupof cells was analyzed separately for different markers (CD40, CD80,CD86, MHCI and MHC II) using the flow cytometer. Theoretically,dendritic cells will engulf antigen and adjuvant and process it in thephagolysosome. The protein fragments will be expressed on MHC II surfacemolecules. For the activation of the CD4+ T cell, a co-stimulatorymolecule known as CD40 is required. CD40 and MHC II expression weresignificantly higher in the VLP-MP+Alum group compared to VLP solution.The inclusion of adjuvant increased CD40 expression and antigenpresentation on MHCII molecules. FIG. 38 is a graph showing theexpression of CD40 on DC 2.4 cells exposed to Blank MP, F-VLP solution,F-VLP MP and VLP MP with adjuvant. MHC II expression was significantlyhigher in MP group compared to VLP solution. CD40 expression was higherin V+Alum group, compared to F-VLP MP group (*p<0.05). Adjuvantsresulted in higher CD40 costimulatory expression. FIG. 39 is a graphshowing the expression of CD80 on DC 2.4 cells exposed to Blank MPs,F-VLP solution, F-VLP MP and VLP MP with adjuvants. CD80 is aco-stimulatory molecule required for activation of CD8 T cells. CD80expression was significantly higher in V MP+Alum (“p<0.01) /MPL A(*p<0.05) group compared to RSV F-VLP MP alone. MF59 did not result inenhanced expression of CD80 molecules. FIG. 40 is a graph showing theexpression of CD40 on DC 2.4 cells exposed to Blank MP, F-VLP solution,F-VLP MP and VLP MP with adjuvant. MHC II is a protein that expressesfragments of antigen to T cells of the immune system. MHC II expressionwas significantly higher in MP group compared to VLP solution. CD40expression was higher in V+Alum group, compared to RSV F-VLP MP group(”p<0.01). Adjuvants resulted in higher CD40 costimulatory expression.

The particles collected were weighed and samples were characterized forsize, surface charge and morphology. The Malvern Zetasizer® Nano ZS wasused to carry out size and surface potential measurements. Particleswere suspended in citric acid for 10 minutes and centrifuged. Theparticles were re-suspended in deionized water and later analyzed by theinstrument. The microparticle images were captured on carbon tape andobserved under 20 kV at 7500× using the Phenom ® Desktop SEM. Thespray-dried microparticles were added to PBS, vortexed for 10 minutesfollowed by incubation at 37° C. to extract the protein antigens fromthe matrix. The sample was centrifuged and supernatant was subjected tothe micro BCA protein assay to quantify the amount of proteinincorporated in microparticles. The vaccine MPs demonstrated immunogenicproperties when compared with its solution counterpart, as seen in FIG.37. We further evaluated the surface marker expression on dendriticcells using the flow cytometer. The vaccine MPs+adjuvants were incubatedwith dendritic cells and incubated for 20 hrs. The cell supernatant wasanalyzed for nitric oxide followed by CD40, CD80, CD86, MHC I and CD54expression which was examined using the flow cytometer. The adjuvantssignificantly increased nitric oxide and surface marker expression ofMHC I and CD80 as seen in FIGS. 37-39. Hence, adjuvants may be effectivein potentiating the immune response.

In Vivo Efficacy of Vaccine Post-Challenge with Live RSV A2 Virus

To evaluate the effectiveness of the F-VLP microparticulate vaccine,mice were dosed using dissolving microneedles. Dissolving microneedles,intended for the painless transdermal release of encapsulatedpharmaceutical agents after dermal insertion, were developed as asolution to the safety issue. Dissolvable microneedles mainly deployPDMS micromolds which are made from a master structure of microneedles(FIG. 5). Briefly, Polydimethylsiloxane (PDMS) was poured onto thestainless steel master structure obtained from Micropoint TechnologiesINC, Singapore (Step 1-3; FIG. 5). The microneedles were made using 10%w/w microparticles, 25% w/w trehalose, 25% w/w maltose, 20% w/wpolyvinylalcohol (PVA), 20% w/w hydroxypropylmethyl cellulose (HPMC).PVA, HPMC, Maltose and Trehalose to a 1.7 mL microcentrifuge tube. Thecontents were dissolved in minimum possible amount of water (e.g. 200 mgof total solid content can be dissolved in 600 uL water) and vortexed.Then approximately ⅕th quantity (of water that was added to dissolve thesolids) of Ammonium hydroxide (NH4OH) was added to the microcentrifugetube (here, 120 uL) and vortexed again. The tube was kept aside for sometime and observed if the contents are dissolved. If everything goes intothe solution, add the weighed amount of vaccine microparticles in theend. This formulation is then added to mold avoiding air bubbles. Thesemolds are then placed straight in 50 mL centrifuge tubes. Centrifugationis done in the fixed angle centrifuge in order to remove air bubbles andto force the formulation to go into the microneedles mold. The maximumspeed is 2000 rpm which is achieved step wise, in order to avoid jerk inthe rotation process, time for which centrifugation should be done is5-10 min. Speed should be lowered gradually for same reason as above.After this centrifugation step, more formulation was added to molds andcentrifugation is repeated in the same manner (Step 3-6; FIG. 5). Thisstep can be repeated further by adding more formulation or blank backinglayer solution. The molds were placed in tubes and placed in anincubator at 37.0 overnight (Step 7-9; FIG. 5).

The groups for the study and doses that were administered to each groupare shown in Table 7.

TABLE 7 Groups for animal study Serial No. Groups VLP/dose Challenge 1PBS (−) Control — RSV-A2 2 IM FI-RSV 1.0 ug FI-RSV RSV-A2 (formalininactivated RSV) 3 Transdermal Suspension 5.0 ug RSV F RSV-A2 (Vaccine +MPL) VLP 4 Transdermal 5.0 ug RSV F RSV-A2 (MP Vaccine) VLP 5Transdermal 5.0 ug RSV F RSV-A2 (MP Vaccine + MPL ®) VLP

For the study, 4-6 week old, male C57BL/6 mice were immunized with 1prime and 2 booster doses of microparticulate RSV F-VLP via thetransdermal route by treating the mice using dissolving microneedles.Blood samples were collected every week and serum antibody (IgG) levelswere analyzed by ELISA. Subsequently, the mice were challenged with liveRSV-A2 virus and body weight was measured for a period of 5 days. At theend of the 5th day, all mice were sacrificed and effector T cellpopulations, specifically, CD4+ and CD8+ T cells were quantified in thelymph node and spleen using fluorescence activated cell sorting (FACS).The lungs were harvested and utilized for histopathology staining andlung viral titer experiments. The lung viral load in the test groupshelped us understand whether the vaccination protocol was effective ingenerating an immune response against the RSV infection.

Experimental Schedule:

FIG. 41 is a timeline for the animal study, with dosing and samplingintervals incorporated.

FIG. 42 is a graph showing IgG antibody levels in blood serum of miceinoculated with Inactivated RSV vaccine (FI-RSV), solution form ofF-VLP, F-VLP microparticles and F-VLP+MPL microparticles. FIG. 43 is agraph showing body weight measurements of mice 6 days post-challengewith live RSV A2 virus. Untreated mice (PBS) showed the highest changein weight compared to vaccinated mice. FIG. 44 and FIG. 45 show graphsof CD4+ and CD8+ T cell response after challenge with live RSV A2 virus.TD MP+MPL showed higher CD4 and CD8 T cell populations compared to TD MPand TD Suspension+MPL. FIG. 46 is a graph of viral titers measured inlung homogenates of various groups after challenge using RT-PCR. Lungviral titers were found to be significantly higher in mice vaccinatedwith inactivated RSV given IM, VLP suspension and VLP microparticlesgiven transdermally compared with VLP MP+MPL A.

EXAMPLE 5 REFERENCES

1. Akalkotkar, A., Tawde, S. A., Chablani, L., & D'Souza, M. J. (2012).Oral delivery of particulate prostate cancer vaccine: in vitro and invivo evaluation. Journal of Drug Targeting, 20(4), 338-346.http://doi.org/10.3109/1061186X.2011.654122

2. Bhowmik, T., D'Souza, B., Shashidharamurthy, R., Oettinger, C.,Selvaraj, P., & D'Souza, M. J. (2011). A novel microparticulate vaccinefor melanoma cancer using transdermal delivery. Journal ofMicroencapsulation, 28(4), 294-300.http://doi.org/10.3109/02652048.2011.559287

3. Chablani, L., Tawde, S. A., Akalkotkar, A., D'Souza, C., Selvaraj,P., & D'Souza, M. J. (2012). Formulation and evaluation of a particulateoral breast cancer vaccine. Journal of Pharmaceutical Sciences, 101(10),3661-3671. http://doi.org/10.1002/jps.23275

4. D'Souza, B., Bhowmik, T., Shashidharamurthy, R., Oettinger, C.,Selvaraj, P., & D'Souza, M. (2012). Oral microparticulate vaccine formelanoma using M-cell targeting. Journal of Drug Targeting, 20(2),166-173. http://doi.org/10.3109/1061186X.2011.622395

5. Dubensky, T. W., & Reed, S. G. (2010). Adjuvants for cancer vaccines.Seminars in Immunology, 22(3), 155-161.http://doi.org/10.1016/j.smim.2010.04.007

6. Kim, H. W., Canchola, J. G., Brandt, C. D., Pyles, G., Chanock, R.M., Jensen, K., & Parrott, R. H. (1969). Respiratory syncytial virusdisease in infants despite prior administration of antigenic inactivatedvaccine. American Journal of Epidemiology, 89(4), 422-434.

7. Kim, K.-H., Lee, Y.-T., Hwang, H. S., Kwon, Y.-M., Kim, M.-C., Ko,E.-J., . . . Kang, S.-M. (2015). Virus-like particle vaccine containingthe F protein of respiratory syncytial virus confers protection withoutpulmonary disease by modulating specific subsets of dendritic cells andeffector T cells. Journal of Virology, JVI.02018-15.http://doi.org/10.1128/JVI.02018-15

8. Li, N., Peng, L.-H., Chen, X., Nakagawa, S., & Gao, J.-Q. (2011).Transcutaneous vaccines: novel advances in technology and delivery forovercoming the barriers. Vaccine, 29(37), 6179-6190.http://doi.org/10.1016/j.vaccine.2011.06.086

9. Murawski, M. R., McGinnes, L. W., Finberg, R. W., Kurt-Jones, E. A.,Massare, M. J., Smith, G., Morrison, T. G. (2010). Newcastle DiseaseVirus-Like Particles Containing Respiratory Syncytial Virus G ProteinInduced Protection in BALB/c Mice, with No Evidence of Immunopathology.Journal of Virology, 84(2), 1110-1123.http://doi.org/10.1128/JVI.01709-09

10. Nair, H., Nokes, D. J., Gessner, B. D., Dherani, M., Madhi, S. A.,Singleton, R. J., Campbell, H. (2010). Global burden of acute lowerrespiratory infections due to respiratory syncytial virus in youngchildren: a systematic review and meta-analysis. Lancet (London,England), 375(9725), 1545-1555.http://doi.org/10.1016/S0140-6736(10)60206-1

11. Quan, F.-S., Kim, Y., Lee, S., Yi, H., Kang, S.-M., Bozja, J.,Compans, R. W. (2011). Viruslike particle vaccine induces protectionagainst respiratory syncytial virus infection in mice. The Journal ofInfectious Diseases, 204(7), 987-995. hap ://doi.org/10.1093/infdis/jir474

12. Rajam, G., Anderton, J. M., Carlone, G. M., Sampson, J. S., & Ades,E. W. (2008). Pneumococcal surface adhesin A (PsaA): a review. CriticalReviews in Microbiology, 34(3-4), 131-142.http://doi.org/10.1080/10408410802275352

13. Shastri, P. N., Kim, M.-C., Quan, F.-S., D'Souza, M. J., & Kang,S.-M. (2012). Immunogenicity and protection of oral influenza vaccinesformulated into microparticles. Journal of Pharmaceutical Sciences,101(10), 3623-3635. http://doi.org/10.1002/jps.23220

14. Ubale, R. V., D'Souza, M. J., Infield, D. T., McCarty, N. A., &Zughaier, S. M. (2013). Formulation of meningococcal capsularpolysaccharide vaccine-loaded microparticles with robust innate immunerecognition. Journal of Microencapsulation, 30(1), 28-41.http://doi.org/10.3109/02652048.2012.692402

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EXAMPLE 6

Encapsulation of Pancreatic Beta Cells for the Treatment of InsulinDependent Diabetes Mellitus

Purpose

This research evaluates the fabrication of microtissue encapsulatingpancreatic islet beta cells (Beta-TC-6) in alginate microcapsules coatedwith chitosan, as therapy for type I diabetes mellitus. Thebiocompatibility and semi-permeable nature of these alginate polymers,in addition to their ability to provide immune protection and their highmechanical properties appears to be a promising strategy for cellencapsulation. In this study, encapsulated cells were evaluated for cellviability, secretion of insulin in the presence of glucose.

Introduction

Diabetes mellitus is a chronic metabolic disease and is one of theprimary causes of mortality in well developed countries. The causativefactor responsible for none or underproduction of insulin is due todestruction of pancreatic beta cells in type I diabetic patients. Thefirst line therapy is to inject insulin directly to patients or eitherorgan transplant. Our strategy is to enclose (encapsulate) pancreaticbeta cells in polymeric microcapsules. This technology works byencapsulating the cells in a semipermeable membrane that allows theentry and exit of small molecules like oxygen and proteins like insulin(Mol wt—6 KDa These cells can produce the protein of interest de novoand deliver the biotherapeutic molecules in the body. The advantage ofour proposed strategy over existing therapy would limit the dosingfrequency and circumvent the need for organ transplantation

Method

As shown in FIG. 47 and FIG. 48, microcapsules were prepared by sprayinga sucrose-alginate-beta cell suspension mixture into calcium chloridesolution using a specialized spray nozzle. Calcium alginatemicrocapsules containing cells were coated with chitosan glutamate toform a semipermeable membrane at the surface. Various concentrations ofsodium alginate, chitosan glutamate and calcium chloride were varied foroptimal size and sphericity.

Procedure:

Briefly, alginate solution (1.2%w/v) was prepared and beta islet cellswere added to it and allowed to stir for fifteen minutes. Afterstirring, the alginate cell suspension was then sprayed via 1.40 mmnozzle using a Buchi spraying apparatus in calcium chloride solution(1.5% w/v). Microcapsules in calcium chloride suspension were allowed tostir for fifteen minutes and washed with PBS, centrifuged twice at ×285g (1200 rpm) to remove the excess calcium ions. Alginate suspension wasthen transferred to chitosan glutamate (0.5% w/v) solution and stirredfurther for fifteen minutes and washed with PBS, centrifuged twice at×285g (1200 rpm) to remove the excess chitosan and finally transferredin DMEM media and kept in the incubator at 37oC.

Results

Microcapsule Size vs Gas Flow Rate

Different sized microcapsules can be achieved by spraying the alginatesuspension at different gas flow rate. Maintaining the higher gas flowrate leads to reduction in size of microcapsules due to high shear atthe tip of nozzle.

Microcapsule Size Distribution

Microcapsule size was measured by using light optical microscope. Thesize distribution was evaluated at mean size of 300 um obtained at a gasflow rate of 250 L/hr. Size distribution was determined by taking atotal of 50 microcapsules. FIG. 49 is a graph of the size ofmicrocapsules (diameter being in μm) plotted at different gas flowrates. Plotted values are mean with ±standard deviation bars. FIG. 50 isa graph of microcapsule size distribution obtained after spraying thealginate suspension at 250 L/Hr.

Representative images of empty microcapsules of size 300 μm are shown inFIG. 51. The microcapsule size was found to be in the range of 200-400μm with a mean of 300 μm.

Fourier Transform Infrared Spectroscopy

FTIR analysis was performed to confirm the cross linking of alginatemicrocapsules with calcium chloride used during the fabrication process.Spectra was taken for powdered samples of sodium alginate and comparedwith the spectra obtained from dried microcapsules. Disappearance ofpeaks in the region of 3250 cm−1 in alginate microcapsules suggestcrosslinking of matrix of alginate microcapsules by calcium chlorideadded during the fabrication of microcapsules as cross linking agent.FIG. 52 is a graph of FTIR spectra of sodium alginate. FIG. 53 is agraph of FTIR spectra of alginate microcapsules.

Light Microscopy Images

Images of microcapsules encapsulated pancreatic islet beta cells weretaken in light optical microscope at 10×. A drop of microcapsulesuspension was placed on slide and coverslip was put on it and observedunder microscope. Also the images were taken at 40× which shows that thebeta cells present inside the microcapsules in small cluster and not assingle cell. The number of cells present in each cluster may range inthousands. FIG. 54A is a light microscopic image showing clusters ofmicrocapsules encapsulated beta islet pancreatic cells were taken atmagnification of 10×. FIG. 54B is a light microscopic image showingclusters of microcapsules encapsulated beta islet pancreatic cells weretaken at magnification and 40×.

Viability of Beta Islet Cells in Microcapsules

Cell viability in the microcapsules is required for the beta cells tosecrete insulin in response to glucose concentration. Cell viability wasmeasured using fluorescent Live/Dead Staining kit. In this assay, theliving cells are stained green by the fluorescent calcein that ishydrolyzed from non-fluorescent calcein AM by the intracellularesterases. Ethidium homodimer-1 (EthD-1) enters only the damaged cellsand yields increased red fluorescence signal upon binding to nucleicacids. Fluorescent images were taken at different time over a period ofthirty days. Stained cells were observed under fluorescent microscopeand photographed with a digital camera.

Procedure—In 96 well plate, 100 μL of sample having microcapsules wereadded to well and 100 μL staining solution (2 mL of 2 mM stock solutionof ethidium bromide and 1 mL of Calcein AM) was added to same well andincubate for 45 minutes. After incubation period, sample was taken onslides and cover with coverslip and analyzed under microscope for liveand dead cells. Cells stained green represents live cells and cellsstained red represents dead cells. FIG. 55 is a chart of Live dead cellstained images of microcapsules encapsulated pancreatic islet beta cellscollected over thirty days period at magnification of 10×.

Nitric Oxide Assay

Nitric oxide is an important marker for innate immune response. Antigenpresenting cells like dendritic cells release nitric oxide upon exposureto an antigen. In this study we found that there is significantly higheramount of nitric oxide released in the supernatant of dendritic cellsexposed to naked beta islet cells compared to microencapsulated cells.FIGS. 56A and 56B are graphs of nitric oxide release vs MC Blank(Microcapsules without beta islet pancreatic cells), MC cells(Microcapsules encapsulate beta islet pancreatic cells and Cells only(Unencapsulated cells) ns-not significant, **p<0.01very significant,***p<0.001 extremely significant.

Stability of Microcapsules

Stability of microcapsules was done for short term (one hour) also knownas explosion assay and long term period (thirty days).

Short Term Stability Studies

Microcapsules fabricated with second layer of coating using chitosanglutamate at different concentrations. Microcapsules were then kept onwater for sixty minutes. Second layer of coating at 1.5 w/v of chitosanglutamate was found showing more than 90% of intact microcapsulessuggesting enhanced stability. However the stability of microcapsulesformed using chitosan glutamate secondary layer at 0.5 W/V and 1.0 W/Vshowed less than 90% of intact microcapsules.

Long Term Stability Studies

For long term stability studies, microcapsules fabricated using chitosanglutamate second layer at 1.5% W/V were kept in DMEM media for a periodof thirty days. Samples were taken at 5 day interval and put of slideswith coverslip and analyzed for percentage of intact microcapsules underlight microscope. It was found that percentage of intact microscope wasmore that 90% on Day 30. FIG. 58 is a graph of long term stabilitymonitored by measuring the fraction of intact microcapsules.

In-Vivo Study

In vivo study was done to evaluate the efficacy of microcapsules instreptozotocin induced diabetic mice model. Animal species used for thestudy was Swiss Webster and mice were considered diabetic when showingblood glucose levels above 250 mg/dL and non-diabetic when blood glucoselevels were below 150 mg/dL.

After the induction of diabetes, mice were segregated into differentgroups based on treatment given. Group received microcapsulesencapsulated pancreatic beta islet cells were injected intraperitoneallywith microcapsules encapsulating beta cells equivalent to 3 millionapproximately. Unencapsulated cells group were injected with cellsequivalent to 3 million cells. Diabetic control group did not receiveany treatment and healthy animals were used in the study for comparisonwith treatment groups. After giving treatment, blood glucose levels weremeasured after every 7 days and for a period of thirty five days. Asshown in FIG. 59, it was found that the blood glucose levels were below150 mg/dL in group received microencapsulated beta cells. However, thegroup received unencapsulated beta cell and diabetic control group showblood glucose level above 250 mg/dL. Blood glucose measurement on Day 42showed blood glucose levels above 150 mg/dL. Therefore, it wasconsidered that the rejection of injected graft took place and no longerefficient in secreting insulin in response to higher blood glucoselevels. FIG. 60 is a Kaplan Miers survival curve shows that themicrocapsule group shows graft rejection on day 42. FIG. 60 showspercent graft survival plotted for different groups i.e., Diabeticcontrol, MC cells (Microcapsules encapsulated pancreatic beta isletcells) and Cells only (Unencapsulated cells).

Body Weight Measurement

Fractional weight was measured for all mice in different groups studiedshown in FIG. 61. Measurement was taken at every 5 day interval andchange in weight of mice was observed during the course of study. Thedata obtained suggest that the increase in weight was higher in groupsadministered with encapsulated cell in microcapsule. However thediabetic control group not received any treatment shows slight increasein weight.

Immunology Study

Immunology study was performed to ascertain the immune response in micereceiving different treatment. After the rejection of graft in micereceived microcapsules encapsulated pancreatic beta cells, mice in allgroups sacrificed and immune organs i.e. spleen and lymph nodes werecollected. Single cell suspension was prepared by passing the spleen andlymph nodes cells through 40μ strainer. Spleen cells obtained weretreated with RBC lysis buffer and centrifuged. This process continuestill the cell suspension obtained was colorless. Then, cells of spleenand lymph nodes were seeded in 48 well plate and incubate with markersof CD4 and CD8 cells. After one hour of incubation, cells were washed toremove excess marker and were analyzed using flow cytometry.

Results of immune study demonstrate that in spleen cells, the CD4expression was higher in microcapsule cell group and it was extremelysignificant (p<0.001) compare to diabetic control group and cells onlygroup. However, the expression of CD8 was higher in cells only groupssuggesting strong immune response and confirms the protection providedthe alginate microcapsules to pancreatic islet beta cells. FIGS. 62A and62B are graphs of percentage of CD4 and CD8 positive cells plotted fordifferent groups i.e., Diabetic control, MC beta and naked Cells Only.*p<0.05 significant, **p<0.01 very significant, ***p<0.001 extremelysignificant (spleen cells). FIG. 62B does not show any statisticalsignificance in expression of CD8 in cells only group and microcapsulegroup but shows scientific significance.

CD4 expression in lymph nodes cells follows the same trend as in spleencells and it was higher in microcapsule cell group and it wasstatistically very significant (p<0.01) in comparison to cells onlygroup as shown in FIGS. 63A and 63B (graphs of percentage of CD4 and CD8positive cells plotted for different groups i.e., Diabetic control, MCbeta and naked Cells Only. *p<0.05 significant, **p<0.01verysignificant, ***p<0.001 extremely significant (Lymph node cells). CD8expression in lymph nodes also follows similar trend as in spleen cellsand it was higher in case of cells only group shows statisticallyextremely significant (p<0.001) in comparison to microcapsule group andtherefore confirms the protective ability of alginate microcapsule tobeta islet cells in response to body immune response.

Expression of CD45R (b220) was also evaluated in order to confiun theantibody response. FIG. 64 is a graph of a flow cytometric analysisshowing CD45R cell counts in different groups of mice *p<0.05significant, **p<0.01very significant, ***p<0.001 extremely significant.FIG. 65 is a graph of a flow cytometric analysis showing CD62L cellcounts in different groups of mice *p<0.05 significant, **p<0.01verysignificant, ***p<0.001 extremely significant. It was found thatantibody secretion similar in all groups and no statistical differencewas found. However, the expression of CD62L, a marker of naïve T cellsshows higher expression in cells only group and statistical verysignificant difference (p<0.01) in comparison to microencapsulated cellsgroup demonstrate less immune response developed for beta islet cellsencapsulated in microcapsules.

EXAMPLE 6 REFERENCES

1. Hong, C., Carol T. B., Sean M. D. C., Adrienne, K. B., Neal N. I.,Collin J. W., and Susan A. S., Long-Term Metabolic Control of AutoimmuneDiabetes in Spontaneously Diabetic Nonobese Diabetic Mice byNonvascularized Microencapsulated Adult Porcine Islets. Transplantation(88) 2 2009 p. 160-169

2. Susan, A. S. Hong, C., M. D., Sean, C., Carol, T. B., B. S., andCollin J. W. M. D. Biocompatibility and Immune Acceptance of AdultPorcine Islets Transplanted Intraperitoneally in Diabetic NOD Mice inCalcium Alginate Poly-L-lysine Microcapsules versus Barium AlginateMicrocapsules without Poly-L-lysine. Journal of Diabetes Science andTechnology (2) 5 2008 p. 760-767

3. Pia, M., Ilaria, P., Teresa, P., Giuseppe, B., Riccardo, C.,Treatment of diabetes mellitus with microencapsulated fetal human liver(FH-B-TPN) engineered cells. Biomaterials (34) 2013 p. 4002-4012

4. Leena, S. K., Marjo, Y., Pyry, T., Antti, M., Ann, M. M., Arto, U., Alaboratory-scale device for the straightforward production of uniform,small sized cell microcapsules with long-term cell viability Journal ofControlled Release (152) 2011 p. 376-381

5. Edward, A. P., Devon, M. H., Robert, T., Peter, M. T., Andres, J. G.,Vasculogenic bio-synthetic hydrogel for enhancement of pancreatic isletengraftment and function in type 1 diabetes. Biomaterials. (19) 2013 p.4602-4611

6. ZHOU, D., SUN, A. M., LI, X., MAMUJEE, S. N., VACEK, I., GEORGIOU,J., WHEELER, M. B., In vitro and in vivo evaluation of insulin-producingbTC6-F7 cells in microcapsules. American Physiological Society 1998 p.C1356-C1362

7. Basta, G., Sarchielli, P., Luca, G., Racanicchi, L., Nastruzzi, C.,Guido, L., Mancuso, F., Macchiarulo, G., Calabrese, G., Brunetti, P.,Calafiore, R., Optimized parameters for microencapsulation of pancreaticislet cells: an in vitro study clueing on islet graft immunoprotectionin type 1 diabetes mellitus. Transplant Immunology (13) 2004 p. 289-296

8. Gasserd, O., Sannes, A., Gudmund, S. B., Microcapsules ofalginate-chitosan. II. A study of capsule stability and permeability.Biomaterials (20) 1999 p. 773-783

9. Jonna, W., Matti, E., Heli, S., Johanna, K., Marjo, Y., Paavo, H.,Arto, U., Alginate-based microencapsulation of retinal pigmentepithelial cell line for cell therapy. Biomaterials (29) 2008 p. 869-876

10. Chris, G., Van, H., Henk, J., BusscherPaul, D. V., Fourier transforminfrared spectroscopy studies of Alginate—PLL capsules with varyingcompositions. Wiley Periodicals, 2003 p. 172-178

11. Min, S. P., Eun, Y. L., Gyo, S., Jacek, W., Andrzej, W., and Hi, B.L., Peritoneal transport of glucose in rat. Peritoneal DialysisInternational (19) 1999 p. 442-450

Although only a number of exemplary embodiments have been described indetail above, those skilled in the art will readily appreciate that manymodifications are possible in the exemplary embodiments withoutmaterially departing from the novel teachings and advantages.Accordingly, all such modifications are intended to be included withinthe scope of this disclosure as defined in the following claims. Unlessotherwise expressly stated, it is in no way intended that any method setforth herein be construed as requiring that its steps be performed in aspecific order. Accordingly, where a method claim does not actuallyrecite an order to be followed by its steps or it is not otherwisespecifically stated in the claims or descriptions that the steps are tobe limited to a specific order, it is no way intended that an order beinferred, in any respect.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.“Exemplary” means “an example of” and is not intended to convey anindication of a preferred or ideal embodiment. “Such as” is not used ina restrictive sense, but for explanatory purposes.

It should further be noted that any patents, applications andpublications referred to herein are incorporated by reference in theirentirety.

Claimed is:
 1. A transdermal delivery system, comprising: (a) at leastone layer of a support material; (b) at least one biodegradable needleassociated with the support material, each needle comprising i. at leastone biodegradable polymer, ii. at least one sugar; wherein eachbiodegradable needle is hollow and is adapted to retain a bioactivematerial.
 2. A method of forming transdeiiiial delivery system,comprising: (a) providing at least one biodegradable polymer material;(b) dissolving the polymer material in a solvent to form a solution; (c)mixing the solution of polymer material of step (b) with at least onesugar to form a polymer-sugar mixture; (d) providing a bioactivematerial; (e) providing a microneedle mold; (f) adding the bioactivematerial and the polymer-sugar mixture of step (c) to the microneedlemold; and, (g) forming at least one microneedle from the polymer-sugarmixture, the at least one microneedle having at least a portion that ishollow, wherein the bioactive material is retained within the hollowportion of the microneedle.
 3. The method of claim 2, further comprisingstep (h) providing a support material and step (i) associating the atleast one microneedle of step (g) with the support material.
 4. Abiodegradable microneedle, comprising: at least one biodegradable needleassociated with the support material, each needle comprising at leastone biodegradable polymer, and at least one sugar, wherein eachbiodegradable needle is at least partially hollow and is adapted toretain a bioactive material.
 5. The system of claim 1, wherein thebiodegradable polymer is polydimethylsiloxane.
 6. The system of claim 1,wherein the sugar is at least one material selected from the groupconsisting of maltose and trehalose.
 7. The system of claim 1, whereinthe bioactive material is at least one material selected from the groupconsisting of drugs, vaccines, and proteins, and mixtures thereof. 8.The system of claim 1, wherein the bioactive material is at least onematerial provided in microencapsulated particle form.
 9. The system ofclaim 1, wherein the at least one biodegradable microneedle is able todegrade within 12 minutes after delivery to a subject's skin.
 10. Thesystem of claim 1, wherein the bioactive material is in the form ofmicroencapsulated material having an average particle size in a range of0.01-50 μm.
 11. The system of claim 1, wherein the bioactive material isin the form of microencapsulated material having an average particlesize in a range of 1-4 μm.
 12. The system of claim 1, further comprisingan adjuvant.
 13. The system of claim 1, wherein the bioactive materialis at least one material selected from the group consisting of a vaccineto gonorrhea, breast cancer, a vaccine to influenza, a vaccine to humanpapilloma virus, and a vaccine to respiratory syncytial virus.
 14. Themethod of claim 2, wherein the biodegradable microneedles areessentially free of organic solvents.
 15. The method of claim 2, whereinthe polymer material comprises polydimethylsiloxane (PDMS).
 16. Themethod of claim 2, wherein the sugar is selected from at least one sugarselected from the group consisting of trehalose and maltose and mixturesthereof.
 17. The method of claim 2, further comprising providingpolyvinyl alcohol (PVA).
 18. The method of claim 2, wherein the solventof step (b) is essentially free of organic solvent material.
 19. Amethod of transdermally delivering a bioactive material, comprising: (a)forming at least one biodegradable and at least partially hollowmicroneedle from at least one biodegradable polymer and at least onesugar; (b) associating a bioactive material with the at least onemicroneedle; (c) associating the at least one microneedle with a backinglayer; and, (d) contacting the at least one microneedle containing thebioactive material with the skin of a subject; whereby the at least onemicroneedle introduces the bioactive material to the subject and the atleast one microneedle biodegrades.
 20. A method of forming transdermaldelivery system, comprising: (a) mixing PVA, HPMC, and the at least onesugar in a vessel; (b) dissolving the mixture of step (a) in water toand mixing to form a mixture; (c) adding ammonium hydroxide to themixture of step (b) and mixing; (d) adding to the mixture of step (c) atleast one bioactive material in microencapsulated form to form aformulation; (e) adding an aliquot of the formulation of step (d) to amicroneedle mold; and, (f) centrifuging the microneedle mold andformulation of step (e) to force the formulation into the microneedlemold.
 21. The method of claim 20, further comprising step (g)associating a backing layer solution with the microneedles.
 22. Themethod of claim 20, further comprising step (h) incubating themicroneedle mold to form a microneedle patch.
 23. The method of claim20, further comprising, after step (f), repeating steps (e) and (f) atleast once.